EUROISMAR 2019

Europe/Berlin
Henry Ford Building

Henry Ford Building

Freie Universität Berlin Garystraße 35 14195 Berlin-Dahlem
Description

Thank you all for your support of EUROISMAR 2019!

Upcoming meetings:

EUROMAR 2020, Bilbao, Spain https://euromar2020.org/

ISMAR-APNMR 2021, Osaka, Japan https://www.ismar-apnmr2021.org/

GDCh FGMR 2020, Bonn, Germany https://www.gdch.de/netzwerk-strukturen/fachstrukturen/magnetische-resonanzspektroskopie.html
Meeting language: English

All the best for the future!

EUROISMAR 2019 is an international conference on magnetic resonance, organized under the auspices of the AMPERE Society and ISMAR. It combines EUROMAR 2019, ISMAR 2019, and the 41st GDCh FGMR annual meeting.

EUROISMAR 2019 will showcase a wide range of research related to the inherent physics of magnetic resonance and its applications in chemistry, biology and medicine. Individual sessions are devoted to new NMR, EPR and MRI methods, applications in material science and biomedical research, dynamic nuclear polarization and other methods for achieving hyperpolarization, quantum computing, in-cell NMR and EPR, as well as applications of low-field NMR, to name a few.

Information for Attendees

Sunday: Registration will take place in the Henry Ford Building.

Organizers

EUROISMAR 2019 acknowledges support from the German Research Foundation.

Contact
• Sunday, 25 August
• 10:00 12:30
Registration 2h 30m
• 12:30 13:00
Luncheon 30m Harnack House

Harnack House

Ihnestraße 16-20, 14195 Berlin
• 13:00 16:00
Bruker User Meeting 3h Harnack House

Harnack House

Ihnestraße 16-20, 14195 Berlin
• 16:00 16:15
Opening of the Meeting 15m Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 16:15 18:55
Prize Lectures: Prize Session 1 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 16:15
Introduction of ISMAR Prize 10m
Speaker: Dr Robert Tycko (National Institutes of Health)
• 16:25
NMR and IDPs 20m

Today we recognize the central role that intrinsically disordered proteins (IDPs) play in cellular processes. This was not always the case. To understand why the IDP concept had not surfaced previously, we need to take ourselves back to the scientific mindset before 1990. The human genome project was not on anybody’s radar – very few if any genes had been sequenced. Cloning and expression of labeled proteins was in its infancy. Computing power was pitiful compared to today – the capacity of our institute-wide mainframe was less than that of a smartphone today. For protein NMR, the dominant technique was all-proton, and 2D was only introduced in 1977. Heteronuclear spectra of uniformly-labeled proteins were introduced in 1989. Our lab was focused on the study of peptides and small proteins, which seemed somehow qualitatively different – peptides were invariably disordered, sometimes with a propensity detectable by NMR for conformational preferences for local structure, whereas proteins were ordered in three dimensions, with a single overall structure that could be reflected in an X-ray crystal structure or, excitingly, by NMR. During the early 1990s we became increasingly puzzled by a series of proteins whose sequences were derived from the earliest sequenced genes. The genetic information was unequivocal on the location of the functional regions of these proteins, but when we looked at them in the NMR, they behaved like peptides. These proteins were disordered, yet still fully functional. Many of them fold into ordered structures upon binding to a partner; many remain fully or partly disordered even in partner complexes. In asking ourselves why disorder might be preferred over order in some types of proteins, we realized that disorder confers important advantages to the efficient operation of cells. Recognition of IDPs would not have been possible without the unique insights provided by NMR.

Speaker: Dr Jane Dyson (Scripps Research)
• 16:45
Relaxing with IDPs: NMR analysis of dynamics and molecular interactions 20m

Intrinsically disordered proteins (IDPs) are highly abundant in the human proteome and are strongly associated with numerous devastating diseases, including cancers, age-related neurodegenerative disorders, diabetes, cardiovascular and infectious diseases. IDPs mediate critical regulatory functions in the cell, including transcription, translation, the cell cycle, and numerous signal transduction events. The lack of stable globular structure confers numerous functional advantages on IDPs, allowing them to exert an exquisite level of control over cellular signaling processes, but poses a major challenge to which traditional structural biology approaches are poorly suited. Many regulatory IDPs contain multiple interaction motifs. The intermolecular interface between such IDPs and their targets is energetically heterogeneous and is characterized by both static and dynamic interactions that mediate crosstalk between signaling pathways and lead to unique allosteric switches. NMR has emerged as the primary tool for elucidation of the structural ensembles, dynamics, interactions, posttranslational modifications, and functional mechanisms of IDPs. Relaxation measurements are especially important for characterization of IDP complexes, providing novel insights into the dynamic processes that mediate binding, competition for a common target, and allostery. The applications of NMR to elucidate the role of IDPs in dynamic cellular signaling will be illustrated by reference to the mechanism of action of a unidirectional, hypersensitive allosteric switch that downregulates the hypoxic response by displacing the hypoxia inducible factor HIF-1α from the general transcriptional coactivators CBP (CREB binding protein) and p300.

Speaker: Dr Peter Wright (Scripps Research)
• 17:10
Introduction of Ernst Prize 10m
Speaker: Prof. Lucia Banci (CERM-University of Florence (Italy))
• 17:20
DEER on cells with Gd(III) 20m

Observing proteins structural changes at during function in-side the cell is a challenge yet to be met. In this context we have been developing in-cell 95 GHz DEER ( double- electron electron resonance) distance measurement using Gd(III) spin labels. The developments that enabled in cell DEER on proteins, including instrumental aspects, Gd(III) spectroscopic properties, the chemistry of the Gd(III) labels and measurement methodology, will be first presented. Next, recent results showing that the monomer- dimer equilibrium constant of a protein is different in vitro and in the cell are different. Similarly, the protein conformation may also be different in these two very different environments.

Speaker: Prof. Daniella Goldfarb (Weizmann Institute of Science)
• 17:40
Everyone needs a little help from one’s friends: Synergy between NMR, cryo-EM and large-Scale MD Simulations 20m

Those of us engaged in active experimental research careers rarely have the opportunity to step back from the lab bench or the computer to reflect on our own scientific practice. Long before descriptive terms such as “multidisciplinary” or “integrative” were fashionable, biological NMR (BioNMR), from its very beginnings, was multidisciplinary in and by itself. Integration of complementary data has a long history in BioNMR and by now everyone in the scientific community is well aware that single types of methodologies are insufficient to adequately describe complex biological structures. I will describe the benefits of integrating solution NMR, MAS solid state NMR, crystallography, cryoEM and large-scale MD simulation, which permitted us to derive a realistic all-atom model for the entire HIV-1 capsid.

Speaker: Prof. Angela M. Gronenborn (University of Pittsburgh School of Medicine)
• 18:10
Introduction of Felix-Bloch Lecture 5m
Speaker: Prof. Burkhard Luy (Institute for Biological Interfaces 4 - Magnetic Resonance and Institute for Organic Chemistry, Karlsruhe Institute of Technology)
• 18:15
Structure and Dynamics of Membrane Proteins in a Native Environment 20m

Membrane proteins are important players in signal transduction and the exchange of metabolites in cells. Thus, this protein class is the target of around 60% of currently marketed drugs, emphasizing their essential biological role. Besides production issues, a major bottleneck encountered in the structural characterization of membrane proteins is identifying a suitable membrane mimetic that provides a native environment. Thus, we are actively developing the phospholipid nanodisc technology for solution-state NMR spectroscopy. This versatile and size-tunable membrane mimetic provides a planar lipid bilayer membrane and can be used to study structure, dynamics and function of integral as well as peripheral membrane proteins. In this talk, our recent advances in the field of nanodisc development will be discussed as well as studies on the structure and dynamics of membrane proteins in suitable membrane mimetics, covering G-protein coupled receptors (GPCRs) and their complexes with G-proteins, a γ-Secretase substrate, the mitochondrial membrane protein VDAC1 as well as its counterpart in chloroplasts, called OEP21. These examples will emphasize the importance of choosing a suitable membrane mimetic for a particular application and membrane protein system.

Speaker: Prof. Franz Hagn (Department of Chemistry and Institute for Advanced Study, Technical University of Munich and Helmholtz Zentrum München)
• 18:55 20:30
Welcome Reception 1h 35m Harnack House

Harnack House

Ihnestraße 16-20, 14195 Berlin
• Monday, 26 August
• 08:40 10:00
Prize Lectures: Prize Session 2 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 08:40
Adventures with Long-Lived States 40m

Long-lived states are particular configurations of nuclear spins which are well-protected against relaxation mechanisms. These configurations appear as particular terms in the spin density operator associated with unusually slow decay rate constants. A seminal example arises in systems of homonuclear spin-1/2 pairs in solution. Singlet order, meaning a population imbalance between the singlet state and the triplet states, is protected against relaxation by the homonuclear dipole-dipole coupling between the two spins, and often has a decay time constant which exceeds T1 by a large factor. In one case, a singlet order lifetime exceeding 1 hour has been observed for a pair of 13C nuclei, even though T1 is about 1 minute under the same conditions.

Our group, and our collaborators, have been enjoying numerous adventures in the world of long-lived states. I will report on some of the following topics:

• using group theory to count and derive long-lived states
• using long-lived states to pump up the nuclear magnetization
• long-lived coherences, including the observation of coherent oscillations proceeding for tens of minutes - even when the sample is removed from the magnet
• relaxation mechanisms for long-lived states, including scalar relaxation in deuterated molecules
• new molecular systems supporting long-lived states
• a new master equation for spin dynamics far from equilibrium
• hyperpolarized long-lived singlet order generated by parahydrogen reactions: geminal-PHIP and trans-PHIP
• spin-isomer conversion of molecules trapped inside fullerenes
Speaker: Prof. Malcolm Levitt (University of Southampton)
• 09:20
New Methods for Dynamic Nuclear Polarization in Insulating Solids: The Overhauser Effect and Time Domain Techniques 20m

Dynamic nuclear polarization (DNP) is now established as a powerful technique for improving the sensitivity of NMR signals by several orders of magnitude, enabling otherwise impossible experiments. Unfortunately, the enhancements obtained at high magnetic fields (> 9 T) are only a small fraction of the theoretical limit due to the fact that current DNP mechanisms, including the cross effect and solid effect, utilize continuous wave (CW) microwave irradiation, and scale unfavorably with B0. This has motivated us to develop new DNP methods that do not suffer from the same field dependences.
Our first attempt resulted in the observation of the Overhauser effect in insulating solids doped with 1,3-bisdiphenylene-2-phenylallyl (BDPA) or sulfonated-BDPA (SA-BDPA) radical. As opposed to all other CW DNP mechanisms, the enhancement of the OE in insulating solids scales favorably with B0. This finding sheds a new light on the seemingly well-understood Overhauser effect.
Our second approach is to perform time domain or pulsed DNP, which differs fundamentally from CW DNP, and like CP and INEPT transfers, is in principle independent of B0. In particular, we have investigated the performance of two related pulse sequences including the nuclear orientation via electron spin locking (NOVEL) and integrated solid effect (ISE) at B0 ranging from 0.35 T to 3.35 T. The NOVEL pulse sequence relies on a matching condition between the nuclear Larmor frequency and the electron Rabi frequency, resulting in a fast polarization transfer from electron to protons (hundreds of ns time scale). Finally, we implemented a new version of the integrated solid effect (ISE) by modulating the microwave frequency instead of sweeping the B0. In comparison to NOVEL, ISE gives similar DNP enhancement even far below the NOVEL condition. Our study sets the foundation for further development of time domain DNP at high fields.

Speaker: Dr Thach Can (Massachusetts Institute of Technology)
• 09:40
Endogenous DNP from Paramagnetic Dopants for Probing Functional Inorganic Materials 20m

In recent years magic angle spinning - dynamic nuclear polarization (MAS-DNP) developed as an excellent approach for boosting the sensitivity of solid state NMR (ssNMR) spectroscopy, thereby enabling the characterization of challenging systems in biology and chemistry. Most commonly, MAS-DNP is based on the use of nitroxide biradicals as polarizing agents. In materials science, since the use of nitroxides often limits the signal enhancement to the materials’ surface and subsurface layers, there is need for hyperpolarization approaches which will provide sensitivity in the bulk of micron sized particles. Furthermore, for many functional materials, e.g. materials used for energy storage and conversion, the use of exogenous polarization agents is limited due to chemical reactivity at the materials’ interface.
Here I will discuss the utilization of paramagnetic metal ion dopants as endogenous DNP agents for sensitivity enhancement in inorganic solids. By introducing the dopants at low concentrations, we obtain NMR signal enhancements of more than two orders of magnitude, thereby enabling the detection of structurally revealing nuclei such as 17O at natural abundance (<0.04%). I will describe the conditions for achieving sensitivity enhancement from a range of paramagnetic dopants and discuss their suitability for probing local vs. remote environments of the dopant. Finally, I will address some of the challenges in implementing this approach in functional solids such as electrode materials for batteries.
The approach offers an alternative route for efficiently detecting reactive surface species and opens the way for structural studies based on high sensitivity NMR of challenging nuclei in the bulk of inorganic solids.

Speaker: Dr Michal Leskes (Department of Materials and Interfaces, Weizmann Institute of Science)
• 10:00 10:30
Coffee 30m HFB Foyer ()

HFB Foyer

• 10:30 13:00
Benchtop & Low-field NMR: Session 1 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 10:30
Advances and Adventures with Compact NMR 35m

My adventures with NMR started in 1973 at the Technical University in Berlin with a just-for-fun project on a 90 MHz ‘high-field‘ Fourier NMR spectrometer [1]. Fun and curiosity has been my main driver since to explore different measurement NMR methodologies along with novel applications. Building on experience with spectroscopy of liquids and solids in homogeneous fields, insights into imaging and the Hahn echo promised new challenges and discoveries with NMR in inhomogeneous fields [2,3]. Such fields can be produced with smaller and cheaper magnets than homogeneous magnetic fields and triggered my quest for the simplest NMR experiment, which lead to stray-field NMR and the NMR-MOUSE. Thanks to my good fortune of working together with brilliant students, postdocs, and colleagues, the NMR-MOUSE evolved into something useful and led to compact magnets with homogeneous fields suitable for NMR spectroscopy. The NMR-MOUSE and benchtop NMR-spectrometers from 40 MHz to 80 MHz are now in the Magritek portfolio, while my group explores the new opportunities provided by their commercial availability [4,5]. This lecture reminisces some of my steps in the world of NMR and reports recent adventures with compact NMR for relaxometry in museums and outdoors as well as for chemical analysis by spectroscopy on the tabletop.

[1] D. Ziessow, B. Blümich, Hadamard-NMR-Spektroskopie, Ber. Bunsenges. Phys. Chem. 78 (1974) 1168-1179
[2] B. Blümich, NMR Imaging of Materials, Clarendon Press, Oxford, 2000
[3] B. Blümich, Essential NMR, 2nd ed., Springer Nature, Cham, 2019
[4] B. Blümich, K. Singh, Angew. Chem. Int. Ed. 57 (2018) 6996 – 7010
[5] B. Blümich, S. Haber-Pohlmeier, W. Zia, Compact NMR, de Gruyter, Berlin, 2014

Speaker: Prof. Bernhard Blümich (RWTH Aachen University)
• 11:05
Understanding novel spin physics to make clinical-scale hyperpolarization simple, fast and cheap 25m

SABRE, a method pioneered about a decade ago, uses parahydrogen and reversible exchange in solution to hyperpolarize organic molecules. In recent years this method has been adapted to polarize heteroatoms such as 15N, rapidly (in seconds) on several hundred different molecules, with an apparatus that can be built for about 1% of the cost a DNP system. Two different strategies have been successful: direct transfer of order from parahydrogen at about 0.5 microtesla (where the resonance frequency difference between nitrogen and hydrogen is about the same as a J coupling) and very weak irradiation of the nitrogen resonance at high field (irradiation strength comparable to a J coupling). Both of these are unusual regimes for magnetic resonance, and provide unique opportunities for exploring spin physics in underexplored domains. This is important because, while progress in a short time has been phenomenal, the fundamental limitation of this technique today is scalability; these methods typically work well at low (mM) concentrations, and not that well in water. New pulse sequences we have developed, coupled with a new quantum Monte Carlo simulation approach, have drastically improved our understanding of the spin dynamics in these complex systems. They have also led to significant signal enhancements, new imaging agents (such as para-nitrogen gas and a variety of injectable compounds) and new strategies for clinical-scale hyperpolarization.

Speaker: Prof. Warren Warren (Duke University)
• 11:30
Compact NMR for Metabolic Health Screening and Diabetes Prevention 25m

The world-wide diabetes pandemic has heightened the need for early screening and prevention. Type 2 diabetes develops slowly and insidiously, and the early stages are often undetected. Here we describe how NMR relaxometry using small table-top devices can be used for the early detection of insulin resistance and metabolic syndrome. These conditions affect nearly one half of US adults and confer a two-to-five-fold increased risk for developing type 2 diabetes.

Water T2, measured using a small volume of unmodified human plasma, serum or whole blood, is exquisitely sensitive to early metabolic dysregulation. In a discovery cohort of asymptomatic non-diabetic human subjects, plasma and serum water T2 showed strong bivariate correlations with markers of insulin, glucose and lipid metabolism, as well as markers of the pro-inflammatory, pro-coagulation state. After correcting for confounding variables using multiple regression, low water T2 values were independently and additively associated with hyperinsulinemia, subclinical inflammation and dyslipidemia, even in subjects with normal glucose levels.

A fingerstick drop of settled whole blood yields two T2 values: one for the plasma supernatant (T2S) and another for the cell pellet (T2P). The T2S value revealed a sixth-power dependence on hematocrit. This sixth-power relaxation enhancement results from a susceptibility gradient from the paramagnetic pellet into the diamagnetic supernatant. Paradoxically, the cell pellet T2P correlated with metabolic markers like those observed with isolated plasma. Here, T2P is sensing modifications in red blood cells resulting from changes in whole body metabolism.

In addition to isolated blood samples, NMR relaxometry measurements can be made non-invasively in living tissues. We will discuss early results obtained using a prototype compact NMR device designed for measuring T2 in the distal segment of the human finger. Compact NMR strategies for monitoring metabolic health are sufficiently practical for translation into point-of-care clinics and community settings.

Speaker: Prof. David Cistola (Texas Tech University Health Sciences Center El Paso)
• 11:55
A MOUSE for Heritage: In Pursuit of Art and Culture 35m

Ancient mummies and bones, paintings and violins – what do they have in common? First, they are all highly relevant to cultural heritage, and second, we can use mobile NMR to learn specific details about each of them.

Mobile NMR is a non-destructive technique that uses single-sided mobile NMR sensors capable of recording NMR signals from samples that are exterior to the magnet. The two main advantages of this method – its portability and non-invasiveness – fulfill the condition for analysis of precious objects that need to be kept safely in museums or archaeological sites and preserved during experiments. This makes mobile NMR an essential tool for studying objects and sites of high interest to the field of cultural heritage.[1,2]

My talk will focus on the applications of the Profile NMR-MOUSE (MObile Universal Surface Explorer) sensor to cultural heritage research. I will present how the NMR-MOUSE can be employed for the characterization of various objects of cultural heritage relevance, ranging from ancient mummies and bones to more recent artifacts, such as older and newer violins as well as modern paintings. I will illustrate how this method can offer information related to the state of conservation of mummies [3], reveal insights into building a master violin and help identifying forgeries in the world of modern paintings.

[1] M. Baias, Mobile NMR: An Essential Tool for Protecting our Cultural Heritage – Magn. Reson. Chem. 55 (2017) 33-37.
[2] M. Baias, B. Bluemich, Nondestructive Testing of Objects from Cultural Heritage with NMR – Modern Magnetic Resonance (2017) 1-13, Springer International Publishing, Ed. Graham A. Webb.
[3] F. Ruehli, T. Boeni, J. Perlo, F. Casanova, M. Baias, E. Egarter, B. Bluemich, Noninvasive spatial tissue discrimination in ancient mummies and bones by in situ portable nuclear magnetic resonance, Journal of Cultural Heritage 8 (2007) 257-263.

Speaker: Prof. Maria Baias (New York University Abu Dhabi)
• 12:30
Wide field range studies of nuclear magnetic relaxation using optically pumped magnetometers 30m

Recently, spin-exchange relaxation free (SERF) alkali-vapor magnetometers have been applied as detectors of nuclear magnetic resonance (NMR) in the zero to ultralow field (ZULF) regime [1]. In ZULF the reduction of spectral line broadening due to field gradients as well as the possible existence of long-lived coherences [2] may lead to spectra with high resolution. These can provide new chemical and physics insight into the sample, beyond the capability of existing analytical techniques. In recent work the technique has been used to provide chemical-specific insight into liquid mixtures after being imbibing into porous catalytic materials [3].
In this presentation, we discuss new methodology that extends the scope of ZULF NMR to study multi-phase materials, including liquids in porous catalytic materials and metals. One aim is to measure 1H NMR relaxation rates T 1 and T 2 at magnetic fields between a few nanotesla and several hundred microtesla, to interrogate slow dynamics associated with surface-site diffusion. These methods are applicable even to materials that cannot be studied with conventional magnetic resonance, including highly paramagnetic, disordered materials.

Speaker: Mr Sven Bodenstedt (ICFO)
• 10:30 13:00
Biomolecules: IDP: Session 4 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 10:30
Disordered Protein Complexes 35m

Intrinsically disordered proteins (IDPs) (or –regions (IDRs)) are functional while existing in broad ensembles of near iso-energetic conformations. Despite their lack of structure, IDPs are involved in molecular communication forming associations ranging from binary, discrete complexes to large multicomponent assemblies. Similar to globular proteins their complexes serve structural, functional and regulatory roles, but due to their dynamic nature, they expand the types of association possible, enabling functional regulations by very different mechanisms. The fast dynamics characteristic of IDPs may persist in their complexes to various degrees. We have been exploring the role of disorder in cellular control processes including pH homeostasis, cytokine signalling, transcriptional regulation, and DNA metabolism, combining NMR with other biophysical methods as SAXS, neutron diffraction, single-molecule FRET and cell biology [1–4]. In one end, we observe folding-upon-binding forming nearly globular-like complexes with little disorder while at the other end, disorder may persist and results in complexes where both binding partners stay disordered in high-affinity binding [3]. Still, the kinetics combined with higher order complex formation allows regulation on biologically relevant timescales. Between these extremes, a continuum of dynamic complexes is possible. The characterisation and functional decoding of dynamic complexes challenges the methodological toolbox, but NMR continues to be a critical contributor in the understanding of disorder dependent biology.

1 Bugge, K., et al (2016) A combined computational and structural model of the full-length human prolactin receptor. Nat Comm 7, 11578.
2 Bugge, K., et al. (2018) Structure of Radical-Induced Cell Death1 Hub Domain Reveals a Common αα-Scaffold for Disorder in Transcriptional Networks. Structure 26, 734–746.
3 Borgia, A., et al. (2018) Extreme disorder in an ultrahigh-affinity protein complex. Nature, 555, 61–66.
4 Hendus-Altenburger, R., et al (2016) The human Na(+)/H(+) exchanger 1 is a membrane scaffold protein for extracellular signal-regulated kinase 2. BMC Biol, 14, 31.

Speaker: Prof. Birthe B. Kragelund (University of Copenhagen)
• 11:05
Cross-correlated relaxation for studying intrinsically disordered proteins 25m

Under physiological conditions intrinsically disordered proteins (IDPs) lack a rigid three-dimensional structure; they can be rather described as a large ensemble of possible structures, that are adopted only transiently [1]. Nonetheless, in organisms they play a variety of roles, e.g. related to signaling and regulation. Interestingly, their flexibility is often crucial for fulfilling these functions. The fact that IDPs are very common, especially in eukaryotic organisms, and their relations to many human diseases, makes them an important object to study.
Due to their relation to backbone dihedral angles, cross-correlated relaxation (CCR) rates are a valuable source of information on protein structure [2]. Such measurements are typically included in three-dimensional (3D) NMR experiments. CCR rates can be also studied for IDPs, where they report on the residual structure. Here, we present the new 4D experiment for the measurement of HNHα diople-dipole – C’ chemical shift anisotropy CCR rate. This rate offers interesting structural information.
The high dimensionality of the proposed experiment provides the resolution that enables efficient studies of IDPs. The inherent flexibility of IDPs causes their chemical shift range to be significantly narrower than in the case of folded proteins. In 3D spectra of an IDP the level of peak overlap can be substantial, limiting the amount of available information. To overcome this problem, the CCR rates measurements can be implemented into higher-dimensional experiments, where the peaks are distributed over a wider spectral space, thus decreasing the number of overlapping peaks [3].
To enable acquisition of high-dimensional data, non-uniform sampling was employed, and for processing a compressed sensing iterative soft thresholding algorithm [4] was used.

References:
[1] Biochimica et Biophysica Acta, 1804(6): 1231-1246, 2010.
[2] Methods in enzymology 338: 35-81, 2002.
[3] Angewandte Chemie – International Edition 52: 4604-4606, 2013.
[4] Angewandte Chemie – International Edition 50: 5556-5559, 2011.

Speaker: Dr Anna Zawadzka-Kazimierczuk (University of Warsaw)
• 11:30
NMR insight into transient structures and interactions within the RNA polymerase of bronchiolitis virus 25m

The RNA polymerase (RNApol) of respiratory syncytial virus (RSV), the main agent responsible for bronchiolitis infections, is a viral RNA synthesis machinery composed of at least of 2 proteins, a 250 kDa catalytic subunit and a 4x27 kDa phosphoprotein co-factor (RSV P). RNApol associates with the viral nucleocapsid, a ribonucleic complex containing the genomic viral RNA matrix, as well as with several other proteins, either viral co-factors or cellular enzymes and signaling proteins. Low affinity towards its partners ensures processivity of the polymerase. Notably many proteins, involved in the RSV RNApol and its complexes display low structural complexity regions, RSV P in particular, which is a linchpin of these complexes. We designed a combination of biochemical and NMR approaches to gain structural and dynamic insight into this protein, which is arranged as a tetramer, but otherwise lacks a defined 3-dimensional structure. NMR revealed structural features ranging from fully disordered regions to ordered regions in the form of isolated helices. This is an advantage for a protein that acts as a platform to recruit specific protein partners either to the RNApol or to replication sites in infected cells. RSV P contains transient secondary structures, which provide plasticity of P and act as contact regions that lead to compaction of the protein or are recognized by protein partners. They may be stabilized in protein complexes. We identified at least four regions that correspond to different partners and complexes. One of these is the RSV M2-1 protein that keeps the RNApol in transcription mode. We also characterized the PP1 phosphatase binding site on P, in the vicinity of that of its substrate, M2-1. Finally, we used NMR for probing protein-protein interaction inhibitors targeting the binding of the nucleocapsid to P, which could pave the way for new antiviral therapeutics.

Speaker: Dr Christina Sizun (CNRS, Institut de Chimie des Substances Naturelles)
• 11:55
The role of proline residues in intrinsically disordered proteins 35m

In recent years many examples of intrinsically disordered proteins (IDPs) appeared in the literature showing how their structural plasticity and intrinsic flexibility enable them to play key roles in many regulatory processes. Their mis-function has also been related to several diseases. The general properties of IDPs cannot be captured in ordered crystals, preventing them to be suitable targets for crystallographic studies. Thus, nuclear magnetic resonance (NMR) spectroscopy plays a crucial role in their investigation, being the only method that allows a high resolution description of their structural and dynamic features in solution.
IDPs are often enriched in the so-called disorder promoting amino acids, among which proline residues have a particular prominence. Proline residues have peculiar physicochemical properties and generally they are not mapped with NMR experiments based on amide-proton detection. Notwithstanding, their role in globular proteins has been studied in detail since decades revealing interesting functional roles. On the contrary, despite their high abundance and presence in many peculiar motifs characteristic of IDPs, the role of proline residues in protein disorder has not yet been addressed in detail.
Examples of proline-rich IDPs will give us the opportunity to discuss the different roles played by proline residues depending on their sequence context.

Speaker: Prof. Roberta Pierattelli (CERM, University of Florence)
• 12:30
The NMR structure of a gp41 cytoplasmic tail fragment reveals the structural basis of the transmembrane coupling of the HIV-1 envelope glycoprotein 30m

The antigenic structure of the HIV-1 envelope spike (Env) is a major consideration for vaccine design to induce effective immune responses. Recent studies suggest that the cytoplasmic tail (CT) of Env influences the antigenic properties of its ectodomain on the opposite side of membrane, but the structural basis of this conformational coupling is still unknown. Using nuclear magnetic resonance (NMR) spectroscopy, we determined the structure in near-lipid bilayer environment of an Env fragment encompassing the transmembrane domain (TMD) and a large portion of the CT, containing the Kennedy sequence (KS) and the lentivirus lytic peptide 2 (LLP2). Structure calculation faced the challenge of constraining the amphipathic CT at the water-lipid interface, which was overcome by implementing plane restraints obtained from NMR-based membrane partition analysis of the protein in bicelles. The structure revealed a molecular architecture never observed before, in which the CT folds into amphipathic helices attaching to the membrane and wraps around the C-terminal end of the TMD, thereby forming a support baseplate for the rest of Env. Biochemical data indicated that the CT-TMD interaction is important for the CT folding and trimerization, which help stabilizing the native conformation of the Env. Functional data from pseudovirus-based neutralization assays confirmed that loosening or disruption of the CT-TMD interaction can indeed affect the antibody binding to the Env ectodomain at the other side of the membrane, as weakening of the CT baseplate can destabilize the conformation of the TMD, which in turn affects that of the membrane-proximal external region (MPER) and the rest of the Env ectodomain, shifting it toward an open conformation. These results contribute to explaining why the CT has a profound effect on the antigenic structure of the ectodomain and can guide HIV-1 immunogen design.

Speaker: Dr Alessandro Piai (Harvard Medical School)
• 10:30 13:00
EPR: Session 2 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 10:30
Pulsed EPR and ENDOR with Photonic Bandgap Superconducting Microresonators 35m

Superconducting microresonators are powerful tools for measuring electron paramagnetic resonance in very small sample volumes. By keeping the thickness of the superconductor below a penetration depth, and aligning the DC magnetic field in the plane of the superconductor, high fields (much larger than the critical field) are possible. With transmission-line geometry resonators (typically coplanar waveguide structures) the mode volume can be a few microns in two dimensions, while of order a wavelength in the third dimension. By utilizing unique properties of superconductors, such as large kinetic inductance, the length of the resonators can be substantially reduced, and planar lumped-element structures can be smaller still. Here we will discuss transmission-line structures which employ "mirrors" consisting of a periodically modulated impedance transmission line. Coplanar waveguide based structures of this variety have a continuous center conductor, allowing DC and low-frequency driving signals, as well as the microwaves for the EPR. A DC current can be used to electrically tune a resonator, using kinetic inductance, though here we will discuss ENDOR experiments in which the RF current is driven through the center conductor. We demonstrate this ENDOR microresonator using phosphorus and arsenic donors in isotopically enriched silicon. Surprisingly, the nuclear spin transitions can also be driven by a resonant electric field (no current) applied to the center pin. For Si:P the effect appears to be mediated by the hyperfine interaction between the donor electron and the nuclear spin. In the case of Si:As, however, the nuclear spins are being driven directly through the nuclear quadrupole interaction. These appear to be the first observations of quadrupolar nuclear electric resonance in a nonpolar crystal.

Speaker: Prof. Stephen Lyon (Princeton University)
• 11:05
Chemical Exchange Processes Studied by 95 GHz 2D-ELDOR 25m

Exchange processes which include conformational change, protonation/deprotonation, binding equilibria etc. are routinely studied by various 2D NMR techniques, e.g. EXSY, ZZ-exchange, CEST. In these techniques the information about exchange of nuclei between environments with different NMR parameters is obtained from the cross-peak development. Cross-peaks due to chemical exchange have been previously seen in EPR, but for most common EPR probes their observation and analysis at low EPR frequencies is difficult because the exchanging states are poorly resolved and their separation is comparable or less than their individual linewidths. With 2D ELDOR spectroscopy at 95GHz we benefited from the increased g-factor resolution to study chemical and physical exchange for protonation/deprotonation and partition equilibria of nitroxide radicals. The protonation/deprotonation process was studied for a pH sensitive imidazoline spin label, with both the relative ratio of exchanging states and the exchange rate controlled by the composition and the concentration of the buffer solution respectively1. This allowed for reliable assignments of cross-peaks related to chemical exchange and for separating them from cross-peaks emerging from Heisenberg exchange and Electron-Nuclear Dipole (END) interactions. The exchange rate obtained from the cross-peaks is in good agreement with the changes in relaxation times of the exchanging states derived from the same 2D ELDOR experiment and other EPR experiments. For a totally different system of a nitroxide radical partitioning between polar and non-polar environments in microemulsions and multilamellar lipid vesicles we also demonstrated the cross-peak development owing to physical exchange between different phases and measured its rate. These experiments were carried out on ACERT’s newly rebuilt 95 GHz 2D ELDOR spectrometer.

1Khramtsov V, Bobko A, Tseitlin M, Driesschaert B. Exchange Phenomena in the Electron Paramagnetic Resonance Spectra of the Nitroxyl and Trityl Radicals: Multifunctional Spectroscopy and Imaging of Local Chemical Microenvironment. Anal Chem. 2017 89(9):4758-4771

Speaker: Dr Boris Dzikovski (Cornell University)
• 11:30
Tuning Spin Dynamics in Crystalline Tetracene 25m

Tetracene is an archetypal material undergoing singlet fission—the generation of a pair of triplet excitons from one singlet exciton. Here, using time-resolved electron spin resonance, we show how the spin dynamics in tetracene crystals are influenced by temperature and morphology. Upon cooling from 300 to 200 K, we observe a switch between singlet fission and intersystem crossing generated triplets, manifesting as an inversion in transient spin polarization. We extract a spin dephasing time of approximately 40 ns for fission-generated triplets at room temperature, nearly 100 times shorter than the dephasing time that we measure for triplets localized on isolated tetracene molecules. These results highlight the importance of morphology and thermal activation in singlet fission systems. In addition, we present recent findings on single crystal tetracene studied using optically detected magnetic resonance, where additional features in the spectrum are observed.

We acknowledge support from DFG SPP-1601 (BI 464/10-2, BE 5126/1-2) and the Nanoscale project within the excellence initiative of the Freie Universität Berlin. The work at Nanjing University is supported by the National Natural Science Foundation of China (21873047). We thank L. R. Weiss for insightful discussions.

Speaker: Dr Naitik A. Panjwani (Berlin Joint EPR Lab, Fachbereich Physik, Freie Universität Berlin, D-14195, Berlin, Germany)
• 11:55
Trityls vs nitroxides as spin labels 35m

Pulse dipolar EPR is widely used to study tertiary structure, dynamics and functional features of biomolecules. Nitroxides are commonly used spin labels. Trityl radicals or TAMs have appeared recently as an alternative source of spin labels using PDEPR[1]. In this presentation we compared functional properties of spin labels based on TAMs and nitroxides.PDEPR in combination with MD were used to investigate the conformational changes in DNA with AP site and in complex with AP endonuclease1 (APE1). For this sake, triarylmethyl (TAM) based spin labels were attached to the 5′-ends of oligonucleotide duplex, and nitroxide spin labels were introduced into the APE1 site. In this way, we for the first time created the system that allowed to follow the conformational changes of the main APE1 substrate by EPR. The use of different (orthogonal) spin labels in the enzyme and in DNA substrate has crucial advantage and allows detailed investigation of local damages and conformational changes in AP-DNA alone, as well as in its complex with APE1. We use very hydrophilic OX063 with very-low toxicity and little tendency for aggregation as the basis for a spin label for human serum albumin (HSA). EPR spectra of HSA-OX063 have an intense, narrow line typical of TAM radicals in solution while HSA-FTAM showed extensive aggregation. In pulse EPR measurements, the measured Tm for HSA-OX063 is 6.3 μs at 50 K, the longest yet obtained with trityl based spin-labels[3]. CW and PDEPR were used to study of the mechanism of penetration of TAM or nitroxide spin labeled intrinsically disordered protein into human cell.

This study was supported by Ministry of Science and Education of the RF(grant14.W03.31.0034).

[1] O.Krumkacheva, E.Bagryanskaya, Trityl radicals as spin labels, From the book: Electron Paramagnetic Resonance:v. 25, 2016,25.
[2] O.Krumkacheva et al.,NAR, 2019, submitted.
[3] V.Tormyshev,et al., Chem. Eur. Jour. 2019, submitted.

Speaker: Prof. Elena Bagryanskaya (N.N.Vorozhtsov Institute of Organic Chemistry SB RAS)
• 12:30
Deep neural network analysis of DEER data 30m

It is demonstrated that deep neural networks (DNN) are a powerful alternative to Tikhonov regularisation methods for the interpretation of double electron-electron resonance (DEER) data. Networks trained using large databases of synthetic DEER traces with carefully modelled distortions and noise are found to process previously unseen experimental data with results comparable to, and occasionally better than, the state-of-the-art Tikhonov methods.

The current best practice for DEER processing is to use Tikhonov regularised deconvolution, a procedure that works well in simple spin-½ pairs, but becomes difficult for more complex systems [1]. Using DNNs trained on simulated data is attractive because training data can be generated to include all complications. Such neural networks can be made resilient to the presence of zero-field splittings, exchange, and out-of-pair inter-electron interactions.

DNN performance strongly depends on the quality of the training dataset. To ensure that the network can successfully process previously unseen datasets, the training database must be representative of the entire range of real experimental systems. The relevant functionality has recently become available in the Spinach library. There are also important factors around the network architecture, pre- and post-processing of data, and the training process.

In this communication we introduce DEERNet and describe how we have created the training database, tuned the network parameters, and handled data pre-processing to achieve excellent performance on real-life DEER data [2].

[1] G. Jeschke, Y. Polyhach, Phys. Chem. Chem. Phys., 2007, 9, 1895-1910.
[2] S.G. Worswick, J.A. Spencer, G. Jeschke, I. Kuprov, Science Advances, 2018, 4 (8), eaat5218.

Speaker: Prof. Ilya Kuprov (University of Southampton)
• 10:30 13:00
In-vivo: Methods: Session 5 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 10:30
Deuterium Metabolic Imaging (DMI), a novel MR-based method for in vivo mapping of metabolism 35m

Deuterium metabolic imaging (DMI) is a novel 3D method to image metabolism of deuterium-labeled substrates in healthy or diseased human brain. DMI provides a powerful tool to reveal altered metabolism and provides completely novel information compared to standard MRI. Examples of aberrant metabolism in aggressive tumors demonstrate the unique metabolic dimension that DMI adds to the standard MRI arsenal. A range of biologically relevant, affordable deuterated substrates are available to probe multiple metabolic pathways. The robustness of DMI together with the high sensitivity, ease-of-use and affordability makes this novel technique highly relevant for 3D mapping of human brain metabolism.

Speaker: Prof. Robin de Graaf (Yale University)
• 11:05
Quantitative Heterogeneity MRS (qhMRS) - A New Type of Line Shape Analysis Applicable to NMR Resonances Sensitive to Suitable Physicochemical Parameters 25m

NMR line shapes have long been analyzed to study coupling constants and multiplicities, molecular structure and mobility, chemical exchange and other molecular properties. Line shape narrowing by complex shim procedures, sample spinning and/or other techniques has been extremely important for optimal suppression of line shape contributions not related to molecular properties. In biomedical in vivo NMR spectroscopy, relatively broad resonances were considered an inevitable annoyance reducing spectral resolution. Indeed, line widths in spectra from biological objects are affected by susceptibility gradients (as a result of tissue structures) that cannot be canceled by shim gradients.

In addition, line shapes of suitable resonances can be characteristically broadened due to specific physicochemical parameters varying across a measured volume of tissue (or other heterogeneous material). A case in point is the shape of the inorganic phosphate (Pi) 31P resonance as its chemical shift is a function of intracellular pH (pHi) [1,2]; or the H2 1H resonance of exogenous imidazole ethoxycarbonylpropionic acid (IEPA) whose chemical shift varies with extracellular pH (pHe) [3]. Here, line shapes are not only broadened, but actually encode information on the statistical distribution of parameter values (pHi or pHe) within the measured pH-heterogeneous sample. We suggest to decode this information by statistical line shape analysis ("quantitative heterogeneity MRS", qhMRS), and present here the experimental proof of principle of our approach, applied to judiciously designed IEPA solutions (phantoms).

This was accomplished by calculating ≥ 8 quantitative descriptors (in addition to the curve maximum) characterizing each statistical distribution of pH values within a given volume or voxel. Based on the H2-IEPA resonance, our qhMRS technique enables integration of statistical pH heterogeneity analysis into 1H MRSI protocols.

1. Lutz NW et al., Cancer Res 2013;73:4616.
2. Graham RA et al., Am J Physiol 1994;266:R638.
3. van Sluis R et al., Magn Reson Med 1999;41:743.
Speaker: Prof. Norbert W. Lutz (CRMBM, Aix-Marseille University)
• 11:30
In-vivo NMR and MRI study of Superparamagnetic Iron Oxide Nanoparticles (SPIONs) on Daphnia Magna 25m

Superparamagnetic Iron Oxide Nanoparticles (SPIONs) are used extensively in multiple biomedical applications such as hyperthermia, MRI enhancing reagents and drug delivery. However, despite their wide spread application, their environmental impacts, especially on the aquatic environment are not well understood. Daphnia magna (water fleas) are keystone species ubiquitous in freshwater ecosystems and are one of the most common species used in toxicity testing. This research combines magnetic resonance imaging, relaxometry and metabolomics (both in-vivo and ex-vivo) to build a holistic understanding of SPION toxicity. SPIONs with three different core sizes (8 nm, 10 nm, 12 nm), and three different ligand sizes (1 KD, 5 KD, 10 KD) were exposed to Daphnia over 24h. The impacts and potential compartmentalization was firstly evaluated by T2 weighted MRI. Secondly, T1 and T2 weighted 2D NMR analyses provided complementary information on the types of biomolecules that interact with the SPIONs inside the organisms. Finally, 2D in-vivo metabolomics provides insight into how the organisms themselves respond on exposure which helps explain the toxic-mode-of-action of the nanoparticles. To our knowledge this is the first time MRI, relaxometry and metabolomics have been combined to provide a comprehensive overview of toxicity inside whole organisms.

Speaker: Dr Bing Wu (University of Toronto)
• 11:55
Imaging Human Brain Metabolism Exploiting Ultra-High Field MRI 35m

In vivo magnetic resonance spectroscopy has evolved during the last 25 years in terms of localization quality and spatial resolution, acquisition speed, artifact suppression, number of detectable metabolites and quantification precision and has benefited from the significant increase of magnetic field strength that recently became available for in vivo investigations. Today it allows for non-invasive and non-ionizing determination of tissue concentrations and metabolic turn-over rates of more than 20 metabolites and compounds with high spatial resolution in the human brain and has established as an important tool for neurophysiological research. This presentation summarizes our recent work using a human 9.4T whole-body MRI scanner. Advantages and technical challenges of ultra-high field human MRI as well as related hardware (RF coils, B0 shimming), data acquisition (RF pulses, Sequences) and data reconstruction approaches are discussed. The high signal-to-noise ratio (SNR) and the spectral resolution at 9.4T in combination with optimized 1H MRSI acquisition and image reconstruction techniques allows for mapping the spatial distribution of a total of 12 metabolites including neurotransmitters, second messengers and antioxidants in the living human brain. Other measurable substances are involved in energy and membrane metabolism. Visualization of concentration differences between gray and white matter and identification of gyri in metabolic MR brain images becomes possible for the first time. In vivo detection of amino acids in vivo is demonstrated and histograms of the amino acid chemical shift distributions extracted from the protein NMR database are used as a fitting model to quantify them. The SNR and spectral separation at UHF also allows for regional functional metabolic readouts and reveal a modulation of energy metabolites and neurotransmitter concentrations. 31P spectroscopy and spectroscopic imaging of the human brain allow mapping of the spatial distribution of energy metabolites such as ATP and NADH. Finally potential neuroscientific and clinical applications are identified.

Speaker: Prof. Anke Henning (University of Texas Southwestern Medical Center)
• 12:30
MRI at 2.15 MHz in a large-bore Halbach Array 30m

Introduction: Modern clinical MRI systems are able to offer sub-millimetre imaging of the human body. However, the high up-front and maintenance cost of these systems means that much of the world lacks access to this technology. For clinical conditions such as hydrocephalus where very high resolution images are not required, low-field MRI systems can offer a low-cost approach towards providing clinically useful MR images in low-resource settings. In this work we designed and constructed a large-bore, low-cost, low-field, Halbach-based MRI scanner intended for neuroimaging in young children.
Methods: The Halbach array is constructed from 23 layers of 12 mm cuboid N48 neodymium magnets, each layer consisting of two rings of magnets with a total magnet length of 50.6 cm with a bore size of 27 cm. The homogeneity of the magnet is improved by tapering the diameter of the Halbach array at the ends. Shimming was performed by placing additional 3 mm cuboid magnets inside the bore of the magnet. Gradient coils were constructed using 1.5 mm diameter copper wire wound on plastic cylindrical formers. A solenoidal RF coil was used for transmit/receive. Total hardware costs were less than 30 000 euros.
Results & discussion: The field strength was 50.45 mT with homogeneity 2500 ppm over a 20 cm diameter spherical volume (DSV). Two dimensional phantom images have been acquired with a resolution of 1x1x35 mm in 16 minutes using a spin echo sequence. Image distortion due to gradient non-linearity was corrected by including simulated gradient fields in a model-based reconstruction.
Conclusion: The homogeneity of a Halbach array can be improved by tapering the diameter of the magnet away from the center. Initial 2D images have been acquired demonstrating the potential for a low-cost MR system.

Speaker: Prof. Andrew Webb (C.J. Gorter Center for High Field MRI, Leiden University Medical Center)
• 10:30 13:00
Solution-state NMR Methods: Session 3 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 10:30
New methods and theory for conformatonal dynamics of macromolecules 35m

Spin relaxation in NMR spectroscopy is a powerful approach for probing aspects of conformational dynamics in biological macromolecules. Methods for characterizing dynamics on picosecond-nanosecond and on microsecond-millisecond time scales, emphasizing the information content provided by multiple static magnetic fields, are illustrated by applications to the enzyme ribonuclease H and the yeast transcription factor GCN4. Theoretical approaches generalize two-state descriptions of the $R_{1\rho}$, Carr-Purcell-Meiboom-Gill, and CEST experiments to N states with arbitrary kinetic topologies, facilitating applications to complex biological phenomena.

Speaker: Dr Arthur Palmer (Columbia University)
• 11:05
Localization of ligands in human carbonic anhydrase by 19F PCS NMR and new lanthanide chelating tags 25m

Pseudocontact shift (PCS) NMR is a powerful tool to gain long-range structural information on protein-protein and protein-ligand interactions. We have shown the localization of three different fluorine containing high affinity ligands within the 30 kDa enzyme human carbonic anhydrase using only 19F PCS NMR [1]. The distances between the 19F atoms and the lanthanide cations ranged between 22 and 38 Å, generating 19F PCS from 0.409 to 0.078 ppm. Up to five different PCS tensors were analysed to extract the fluorine positions with an accuracy of up to 0.8 Å. A careful investigation on the influence of the number and orientation of the tensors for the different ligands on the precision of the localization revealed new insights into the potentials and limits of PCS NMR, especially for ligand screening.
We would like to report in addition on several new lanthanide chelating tags (LCT), based on sterically overcrowded tetra-methyl- or tetra-isopropyl-cyclen scaffolds.
Combination with bulky side-arms and a highly reactive, reduction stable pyridine-thiazolo linker results in versatile LCT. All new tags were benchmarked on ubiquitin and also on human carbonic anhydrase and demonstrated very large PCS and RDC. In order to explore the theoretical PCS limit for an LCT with ideal rigidity relative to the protein, we determined for the first time the intrinsic anisotropy parameters of strongly paramagnetic LCT on the free tags. To assign the proton NMR spectra that display a proton chemical shift range of up to 1500 ppm and a T2 relaxation time of less then 50 µs, various isotope labelling schemes had to be used, as 2D NMR spectra are not feasible. Besides unusually large susceptibility tensors, we also unravelled Fermi contact shifts in the proton spectra that are in contradiction to current theoretical models.
Ref: [1] K. Zimmermann et al., Chem. Sci. 2019, DOI: 10.1039/c8sc05683h

Speaker: Dr Daniel Häussinger (University of Basel)
• 11:30
Accurate Measurement of Transverse Relaxation Rates in Systems with Coupled Protons 25m

Measuring transverse relaxation rates provides insight into the dynamics of molecules. For example, measurements of relaxation dispersion allow the study of invisible conformations of proteins. Such applications are currently restricted to sparse spin systems in which homonuclear couplings can be neglected, such as 15NH or selectively labelled 13CHD2 groups in proteins. The accurate measurement of transverse relaxation rates is also essential if quantitative results are to be obtained from multipulse NMR experiments where there are T2 differences among the spins observed.
The common experiments for measuring T2 in coupled spin systems, CPMG [1] and PROJECT [2], can suppress the signal modulations caused by homonuclear J couplings. However in both cases the relaxation measured is not specific to a given signal, but contains contributions from all the spins in a coupled system because of the sharing of coherence. Measured T2 values therefore differ from the true T2s. We propose a new approach, Active Spin Refocusing (ASR) T2 measurement, which allows broadband measurement of ‘true’ transverse relaxation rates even in coupled spin systems. It uses a single spin echo, with the refocusing element flanked by variable delays instead of trains of pulses so that the measured signal decay reflects the true loss of transverse magnetisation during free precession. J modulation is suppressed by using an active spin refocusing element of the sort now commonly used in pure shift NMR methods such as that of Zangger and Sterk [3]. This allows true T2 measurements on all signals in a spectrum in a single experiment, albeit at the price of a reduction in sensitivity.

References
[1] S. Meiboom and D. Gill, Rev. Sci. Instr., 1958, 29, 688-691.
[2] J.A. Aguilar et al, Chem. Commun., 2012, 48, 811-813.
[3] K. Zangger and H. Sterk, J. Magn. Reson., 1997, 124, 486-489.

Speaker: Dr Peter Kiraly (University of Manchester)
• 11:55
Conformation Changes in Proteins Made Visible by Lanthanide Tags 35m

The coordinates of a nuclear spin relative to a lanthanide ion can be determined with high accuracy in a system, where a paramagnetic lanthanide tag is attached to a protein of known three-dimensional structure. First, pseudocontact shifts (PCS) of the protein are measured by NMR to determine the coordinate frame of the magnetic susceptibility anisotropy (DeltaChi) tensor associated with the lanthanide tag. Next, the PCS of the nuclear spin of interest is used positions it relative to the DeltaChi tensor. With lanthanide tags at three different sites, the position of the nuclear spin can be restricted in a way analogous to the global positioning system (GPS) of a mobile phone. Examples of this approach are shown for changes in amino acid side chain position triggered by metal binding and in response to ligand binding.

Speaker: Prof. Gottfried Otting (Australian National University)
• 12:30
De-correlating kinetic and relaxation parameters in exchange saturation transfer NMR 30m

The interaction of the N-terminal domain of huntingtin exon-1 with membrane surfaces promotes poly-glutamine mediated aggregation, and is thought to play a role in the etiology of Huntington’s disease. We investigated the kinetics of binding of two huntingtin peptides, comprising the 16-residue N-terminal amphiphilic domain alone (httNT) and with a seven residue poly-glutamine C-terminal tract (httNTQ7), to small unilamellar lipid vesicles (SUV), ~31 nm in diameter and ~4.3 MDa in molecular weight, using solution NMR experiments designed to probe interactions of NMR visible states with sparsely-populated, invisible or 'dark', high molecular weight species. Specifically, we make use of Dark state Exchange Saturation Transfer (DEST) and lifetime line broadening (dR2) supplemented with the measurements of the maximal value of the contribution of fast-relaxing magnetization component to the total NMR signal, Cfast_max. In the exchange regime where the transverse spin relaxation rates in the bound state are smaller than the strength of the DEST saturation radio-frequency field, the combination of DEST and dR2 data is not sufficient to unambiguously determine the population of the bound state (pB) and its transverse relaxation rates at the same time. We show that these exchange and relaxation parameters can be de-correlated by the measurement of Cfast_max which is directly proportional to pB. When integrated into the analysis of DEST/dR2 data, Cfast_max provides an indispensable source of information for quantitative studies of exchange involving high-molecular-weight dark states. While the population of the species bound to the SUV surface is substantial, on the order of 7-8%, the exchange between the free peptides and the SUV-bound states is slow on the relaxation time-scale (kex~200 1/s). The C-terminal regions of the peptides remain flexible even in the SUV-bound form due to transient detachment from the lipid surface that occurs on a time-scale several-fold faster than the binding proper.

Speaker: Dr Vitali Tugarinov (National Institutes of Health)
• 13:00 14:00
Lunch 1h Harnack House

Harnack House

• 14:00 16:00
Posters: odd numbered presentations Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

• 16:00 16:15
Coffee 15m Harnack House

Harnack House

• 16:15 17:40
Biomolecules: Carbohydrate Interactions: Session 9 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 16:15
Selective High-Resolution DNP-Enhanced NMR of Biomolecular Binding Sites 35m

Locating binding sites in biomolecular assemblies and solving their structures is crucial to unravel functional aspects of the system and provide experimental data that can be used for structure-based drug design. This often still remains a challenge, both in terms of selectivity and sensitivity for X-ray crystallography, cryo-electron microscopy and NMR.
Dynamic Nuclear Polarization (DNP) has revolutionized the scope of many solid-state NMR experiments by enabling new sensitivity-limited experiments to be recorded. Its use for biomolecular systems is however often limited by the necessity to run experiments at cryogenic temperatures, which can induce line broadening and loss of resolution. We present here a new method, called Selective DNP (Sel-DNP) that provides specific selectivity with high spectral resolution for the binding region of biomolecules, allowing for the identification of the residues present in the binding site. This powerful site-directed approach relies on the combined use of localized paramagnetic relaxation enhancement, induced by a ligand-functionalized paramagnetic construct, and difference spectroscopy to recover high-resolution and high-sensitivity information from binding sites.
The Sel-DNP approach is demonstrated on the galactophilic lectin LecA, a 12.75 kDa protein. The identification of residue types present in the galactose-binding region occurs using spectral fingerprints obtained from a set of high-resolution multidimensional spectra with varying selectivity. Experimental and computational strategies are then combined to assign the identified residues types to the specific residues in the sequence of the protein. In particular, a hierarchical alignment procedure using a modified genetic algorithm will be presented, which is able to perform de novo assignment and to locate the binding site in the protein sequence on the sole basis of the residue-type list extracted from Sel-DNP spectra.

Speaker: Dr Sabine Hediger (CNRS)
• 16:50
Interaction between Cell-Wall and Biosynthetic Enzymes Using a Combination of Liquid- and Solid-State NMR Approaches 25m

The cell wall is essential for the survival of bacteria. It gives the bacterial cell its shape and protects it against osmotic pressure, while allowing cell growth and division.
The machinery involved in the synthesis of this envelop is crucial and is one of the main antibiotic target. Different proteins as transpeptidases, transpeptidase activators or hydrolases are recruited to maintain the morphogenesis of this polymer during the bacterial cell cycle. Based on few examples involved in the machinery of synthesis of the peptidoglycan, we will present a combination of liquid and solid-state NMR that can be a powerful tool to screen for cell-wall interacting proteins in vitro and on cell.
In particular, we have explored the possibilities to study the PG with ultra-fast (100 kHz) magic-angle spinning NMR. We show that highly resolved spectra can be obtained, and we have developed strategies to obtain site-specific resonance assignments and distance information. we have also in parallel investigated the potential of Dynamic Nuclear Polarization (DNP) to investigate cell surface directly in intact cells.
Altogether, NMR approaches developed here propose new routes to fill the gap between in vitro studies of isolated biomacromolecules and in vivo cell biology studies in order to investigate cell surfaces and decipher key biological processes involved.

Speaker: Prof. Jean-Pierre Simorre (CNRS)
• 17:15
Unravelling Glycan-Lectin Interactions: from STD to Paramagnetic NMR 25m

Molecular recognition by specific targets is at the heart of the life processes. In recent years, it has been shown that the interactions between proteins (lectins, enzymes, antibodies) and carbohydrates mediate a broad range of biological activities, from fertilization and tissue maturation, to pathological processes. The elucidation of the mechanisms that govern how sugars are accommodated in the binding sites of these receptors is currently a topic of interest. Thus, unravelling the structural and conformational factors and the physicochemical features that rule the interactions of these molecules is of paramount interest. The key tool for studying at atomic resolution the recognition processes in which glycans are involved is NMR. Thus, we use NMR as key tool for analysing key molecular recognition processes in which glycans are involved at atomic resolution.[1-8] Although the inherent flexibility of N-glycans and the chemical equivalence of individual branches precludes their NMR characterization using standard NMR methods, using multi-antennary N-glycans conjugated to a lanthanide binding tag, we have been able to discriminate the NMR signals of bi and multiantennary glycans with unprecedented resolution. As recent example, key details of biantennary glycan recognition by influenza hemagglutinin will be shown, with special emphasis in the application of novel paramagnetic-NMR methods to evaluate the relative importance of polar (hydrogen bonding, electrostatic interactions) and non-polar (van der Waals, CH-π) forces in the recognition process.

1. A. Gimeno, et al. ACS Chem Biol. 2017, 12, 1104.
2. L. Unione, et al., Chem Eur J. 2017, 23, 3957.
3. A. Canales, et al., Angew Chem Int Ed. 2017, 56, 14987.
4. B. Fernández de Toro, et al., Angew Chem Int Ed. 2018, 57, 15051.
5. T. Diercks et al., Chem Eur J. 2018, 24, 15761.
6. A. Ardá, J. Jiménez-Barbero, Chem Commun. 2018, 54, 4761.
Speaker: Prof. Jesus Jimenez-Barbero (CIC bioGUNE)
• 16:15 17:40
Biomolecules: Membranes: Session 8 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 16:15
New Long-Distance SSNMR Techniques and Their Applications to Protein Structure Determination 35m

In this talk I will first present our recent development of multiple 19F-based solid-state NMR techniques that increase the distance reach of NMR to ~2 nm. 19F-19F distances can be measured by both spin diffusion and dipolar recoupling techniques under fast MAS frequencies of 25 – 55 kHz at the medium-high magnetic field of 14.1 Tesla. Fast MAS suppresses the 19F chemical shift anisotropy sidebands without compromising the dipolar coupling measurements. An empirically calibrated master curve has been obtained from 19F-19F spin diffusion buildup rates to allow extraction of semi-quantitative distances. We also demonstrate 13C-19F and 1H-19F distance measurements under fast MAS. These long-distance 19F SSNMR techniques are instrumental for determining the cholesterol-binding site of the influenza M2 protein, the oligomeric structure of the HIV fusion protein gp41, and the interhelical packing of the influenza B virus M2 protein. In the second part of the talk, I will describe the determination of a novel amyloid fibril structure formed by the anti-hypoglycemia pharmaceutical peptide, glucagon.

Speaker: Prof. Mei Hong (MIT)
• 16:50
Molecular mechanisms behind Remorin nanodomain formation 25m

Protein and lipid components in biological membranes act as a dynamic network of subtle molecular interactions segregating the membrane into particular highly dynamic regions called nanodomains. Nanodomains constitute functional platforms enriched in specific lipids (such as sterols and phosphoinositides) and proteins to perform their diverse activities. Remorins (REMs) are plant proteins and well-established nanodomain markers and can, as such, be considered as paradigm to provide a mechanistic description of membrane organisation into functional nanodomains. In a divide-and-conquer approach, we describe the impact of StREM1.3’s C-terminal membrane anchor (1), its oligomerization domain (2) and the intrinsically disordered region on membrane structure and dynamics. Furthermore, we tackle the structural features of StREM1.3 and its domains when associated to nanodomain-mimicking membranes by solid-state NMR. We show that StREM1.3 drives nanodomain organisation by concerted lipid-protein and protein-protein interactions, highlighting the dedicated role of each domain. We reveal a delicate balance between hydrophobic and electrostatic effects leading up to the protein’s characteristic affinity for negatively charged phospholipids.

(1) Gronnier J, et al.; Elife. 2017; 6. pii: e26404
(2) Martinez D, et al.; J Struct Biol. 2019; 206(1); 12-19

Speaker: Dr Birgit Habenstein (CBMN / IECB UMR 5248 CNRS University of Bordeaux)
• 17:15
Solution NMR of nanodisc-embedded proteins: new molecular insights into protein-protein and protein-membrane interactions 25m

Membrane-associated proteins (MAPs), such as channels, pumps and receptors, are notoriously difficult to study by structural methods because they require a stabilizing surrogate lipo-environment, often making sample preparation and data acquisition challenging. At the same time biological processes occurring at the cell membrane, particularly protein-protein and protein-membrane interactions, are deeply involved in homeostasis and disease; indeed, over 50% of approved pharmaceuticals target this class of cellular contacts. Thus, there is great motivation to reach a structural understanding of membrane protein biochemistry despite these objective challenges. Here we demonstrate successful applications of the lipoprotein nanodisc (LPN) technology, providing close-to-native membrane assemblies, to addressing structural questions in the membrane environment.

By incorporating the potassium channel KcsA in LPNs we could reliably identify the molecular basis of biological function in two regions of interest, the toxin-binding extracellular region and the putative pH-gating region in the cytoplasmic C-terminal domain. We used NMR to determine the structures of de novo and natural KcsA-blocking toxins, and, in combination with electrophysiology measurements, the basis for specific recognition between toxins and various channels. LPNs also enabled us to follow the tetramer-to-monomer transition in the channel’s cytoplasmic domain by NMR and EPR in terms of pH-gating, a subject of controversy in previous studies, as well as coupling to other gates. Finally, we employed LPNs to investigate host membrane-targeting by the cytotoxic effector BteA, secreted by the pathogen Bordetella pertussis responsible for causing whooping cough. Chemical shift perturbation analysis of wildtype and mutant BteA, backed by additional biophysical methods, showed that this four-helix bundle domain binds to membranes in a phosphatidylinositol-dependent manner, and defined a membrane-targeting motif that differs from that of previously described effectors. We thus demonstrate the utility of NMR methods in conjunction with LPNs in elucidating the structural biology of membrane-associated proteins.

Speaker: Prof. Jordan Chill (Department of Chemistry, Bar Ilan University)
• 16:15 17:40
Biomolecules: Modelling of Biological Processes: Session 7 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 16:15
Copper trafficking in eukaryotic systems: Current knowledge from experimental and computational efforts 35m

Copper plays a vital role in fundamental cellular functions, and its concentration in the cell must be tightly regulated, as dysfunction of copper homeostasis is linked to severe neurological diseases and cancer. This talk provides a compendium of current knowledge regarding the mechanism of copper transfer from the blood system to the Golgi apparatus; this mechanism involves the blood carrier protein, human serum albumin (HSA), the copper transporter hCtr1, the metallochaperone Atox1, and the ATPases ATP7A/B. I will discuss key insights regarding the structural and functional properties of the hCtr1-Atox1-ATP7B cycle, obtained from diverse studies relying on Electron Paramagnetic Resonance (EPR) measurements, complementary biophysical methods, biochemical, and computational methods. Last, I will demonstrate how basic under-standing of the function of these systems can assist us in designing new class of bi-markers and therapeutic agents.

Speaker: Prof. Sharon Ruthstein (Bar Ilan University)
• 16:50
Structural description of the target search process by a disordered transcription factor 25m

Specific transcription factors must search for their target sites amongst a vast excess of non-specific DNA. They find their sites quickly using a combination of sliding, jumping, hopping and intersegmental transfer. DNA binding domains (DBDs) are usually thought of as structured, however this is not always the case. There are also 3 very large classes of transcription factors whose DBD are disordered in the absence of DNA – bZIPs, bHLH and AT hooks. I have been examining a prominent model protein, cyclic-AMP response element binding (CREB) protein, a member of the bZIP family, to determine what role protein disorder might play in the target search process. CREB binds to its target as a homodimer. We present structural data from NMR for both monomeric and dimeric CREB that describes its secondary structure propensity and dynamics when free in solution, sliding along the DNA and bound to its target site. We demonstrate that whilst searching for its target site the protein remains highly dynamic with limited helical content, and that the protein forms a dimer before binding to its target site. To our knowledge our results constitute the most complete structural description of the search process by a disordered transcription factor to date.

Speaker: Mr Conor Kelly (University of Oxford)
• 17:15
A generalized approach for NMR studies of lipid–protein interactions based on sparse fluorination of acyl chains 25m

High-resolution NMR studies on protein-lipid interactions are severely limited by poor 1H signal dispersion in the lipids' acyl chains, where uniform 13C enrichment cannot resolve all overlap problems and introduces no distinct molecular marker from a likewise 13C enriched protein to separate inter- from intramolecular NOE signals. We present a new approach [1] that relies on sparse fluorination of lipid acyl chains and exploits fluorine both indirectly, as a shift reagent affecting nearby spins, and directly, as a distinctive isotope (19F) with superb NMR properties. The introduced fluorine atoms then solve the NMR resolution problem for acyl chains by (i) increasing their 1H signal dispersion via local deshielding, (ii) enabling clean molecular distinction via 19F filtering, and (iii) allowing further resolution enhancement via 19F editing. While the number of H/F substitutions must be minimised to mitigate any biophysical impact, prevent complications from 1JFF coupling, and preserve a high 1Hlipid density to probe intermolecular contacts via 1Hlipid-1Hprotein NOE signals, a minimal fluorination scheme is defined by the reach of fluorine induced deshielding and JHF coupling. We, thus, designed di-(4-fluoro)heptanoyl¬phosphocholine (4F-DHPC7) that forms stable micelles with similar size as DHPC7, but with fully dispersed high-resolution 1H and 19F spectra. Both DHPC7 and 4F-DHPC7 micelles readily stabilise the phototaxis receptor sensory rhodopsin II (pSRII) and outer membrane protein X (OmpX), where 15N TROSY signals differ notably only for residues near the fluorine atoms in modelled 4F-DHPC7 micelles. Thus, H/F substitution in lipid chains also causes localised fluorine induced chemical shift perturbations (CSPF) in solubilised proteins, indicating their lipid layer insertion similar to paramagnetic markers, but with minimal steric impact and finer distance resolution. Finally, a first 19F filtered NOESY spectrum unambiguously brought out intermolecular contacts between 4F-DHPC7 and a bihelical integrin fragment.
[1] De Biasio et al, Chem.Comm. (2018) 54, 7306-7309

Speaker: Dr Tammo Diercks (CiC bioGUNE)
• 16:15 17:40
Materials: Session 6 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 16:15
Probing Ion Mobility Mechanisms in Solid Electrolytes using Solid-State NMR 35m

All-solid-state Li-ion batteries are attracting considerable attention as possible alternatives to conventional liquid electrolyte-based devices as they present a viable opportunity for increased energy density and safety. In recent years, a number of candidate materials have been explored as possible solid electrolytes, including garnets, Li-stuffed garnets, Li-rich anti-perovskites (LiRAPs), thio-LISICONs and complex spinels. LiRAPs, including Li3−xOHxCl, have generated considerable interest based on their reported ionic conductivities (on the order of 10−3 S cm−1).1,2 However, until very recently, their lithium and proton transport capabilities as a function of composition were not fully understood. Hence, current research efforts have focused on the synthesis and structural characterisation of Li3−xOHxCl using a combination of ab initio molecular dynamics and variable-temperature 1,2H, 7Li and 35Cl solid-state NMR spectroscopy. Using this unique combination of techniques, it is possible to study the mobility of both the Li ions and protons. We will demonstrate that Li-ion transport is highly correlated with the proton and Li-ion vacancy concentrations. In particular, we will show that the Li ions are free to move throughout the structure, whilst the protons are restricted to solely rotation of the OH groups. Based on these findings, and the strong correlation between long-range Li-ion transport and OH rotation, we have proposed a new Li-ion hopping mechanism, which suggests that the Li-rich anti-perovskite system is an excellent candidate electrolyte for all-solid-state batteries.3 However, to fully understand the mechanism for conduction, multiple, complementary characterisation techniques are needed.

References

1. Y. Zhao and L. L. Daemen, J. Am. Chem. Soc., 2012, 134, 15042.
2. A. Emly, E. Kioupakis and A. Van der Ven, Chem. Mater., 2013, 25, 4663.
3. J. A. Dawson, T. S. Attari, H. Chen, S. P. Emge, K. E. Johnston and M. S. Islam, Energy Environ. Sci., 2018, 10, 2993.
Speaker: Dr Karen Johnston (Durham University)
• 16:50
Structure and Dynamics of Defects in Metal-organic Frameworks studied by Solid-state NMR 25m

Metal-organic frameworks (MOFs) are porous crystalline materials with promising applications in molecular adsorption, separation, and catalysis. It has been discovered recently that structural defects introduced unintentionally or by design could have a significant impact on their properties. However, the exact chemical composition and structural evolution under different conditions at the defects are still under debate.

In the first part, we probed the existence of residual modulator: the commonly-used acetic acid, which controls the formation of defects in UIO-66. We discovered that acetate molecules coordinate to a single metal site monodentately and pair with water at the neighboring position. The acetates are highly flexible which undergo fast libration as well as a slow kinetic exchange with water through dynamic hydrogen bonds. The dynamic processes under variable temperatures and different hydration levels have been quantitively analyzed by SUPER and CODEX experiments. The integration of SSNMR and computer simulations allow a precision probe into defective MOF structures with intrinsic dynamics and disorder.

In another related work, we studied Mg2(dobpdc) (dobpdc4− = 4,4′- dioxidobiphenyl-3,3′-dicarboxylate) that contains accessible coordinatively unsaturated metal sites. We investigated the defect chemistry of Mg2(dobpdc) when synthesized with 4-fluorosalicylic modulators. We illustrated that by varying the concentration of modulator, the linker vacancies can be tuned systematically and the concentration of the ligand substitution defects can be as high as ∼35%. We uncovered the detailed structure of modulated Mg2(dobpdc) and the defects distribution by REDOR solid-state NMR experiments.

Y. Fu, Z. Kang, J. Yin, W. Cao, Y. Tu, Q. Wang, X. Kong Nano Letters, 2019, 19.1618-1624

Speaker: Ms Yao Fu (Zhejiang University)
• 17:15
Solid-state and in situ NMR studies of flexible metal-organic frameworks 25m

Framework flexibility (elasticity), i.e., the ability of a metal-organic framework (MOF) to considerably change its structure as a function of relevant parameters like pressure, temperature, and type of adsorbed molecules is only observed for some special compounds. The MOF compound Ni2(2,6-ndc)2(dabco) [2,6-ndc: 2,6-naphthalenedicarboxylate, dabco: 1,4-diazabicyclo[2.2.2]octane, further denoted as DUT-8(Ni)] can be synthesized in flexible and non-flexible (rigid) form of equal chemical composition just by controlling the particle size [1]. The unit cell of the flexible form changes its volume by more than 100% during the reversible, adsorption-induced structure opening! Here, we comparatively study flexible and non-flexible DUT-8(Ni) in order to answer two questions: What are the structural differences between these two variants? And does framework flexibility influence adsorption selectivity from gas mixtures? To answer the first question, we selectively isotope-labeled promising framework positions with 13C and 2H. This allows to selectively detect carboxylate 13C atoms close by the Ni centers and to study the mobility of the organic linker molecules by 2H NMR spectroscopy. Extended solid-state NMR investigations encompassing 2H exchange spectroscopy (EXSY), 13C-1H heteronuclear correlation (HETCOR), and others revealed that the non-flexible form exhibits a higher fraction of defects and dynamically disordered linker molecules compared to the flexible variant. The second question is studied by quantitative high-pressure in situ 13C NMR spectroscopy of gas adsorption from mixtures containing 13C-enriched CO2 and CH4. Flexible DUT-8(Ni) indeeed exhibits a significantly higher selectivity for carbon dioxide adsorption from these mixtures than the rigid form [2]. That means, framework flexibility seems to influence adsorption selectivity.

[1] N. Kavoosi, V. Bon, I. Senkovska, S. Krause, C. Atzori, F. Bonino, J. Pallmann, S. Paasch, E. Brunner, S. Kaskel, Dalton Trans. 2017, 46, 4685.
[2] M. Sin, N. Kavoosi, M. Rauche, J. Pallmann, S. Paasch, I. Senkovska, S. Kaskel, E. Brunner, Langmuir 2019, 35, 3162.

Speaker: Prof. Eike Brunner (TU Dresden)
• 16:15 17:40
Spin Physics: Session 10 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 16:15
Electron Spin Relaxation Mechanisms of Atomic Hydrogen Trapped in Silsesquioxane Cages: the Role of Isotope Substitution 35m

Encapsulated atomic hydrogen in silsesquioxane cages is a promising candidate for applications in emerging technologies like spin-based quantum computing, magnetic field sensing, and atomic clock devices. Compared to endohedral fullerenes (N@C$_{60}$ or P@C$_{60}$), which are currently the most used molecular spin systems for demonstrating single-quantum gates and quantum memories, atomic hydrogen is more attractive due to its simpler electronic 1s state and the exceptionally large hyperfine coupling of about 1420 MHz. Detailed pulsed EPR studies of parameters relevant to quantum computing like electron spin-lattice ($T_1$) and phase memory ($T_M$) relaxation times are scarce and concern exclusively cages of the type Si$_8$O$_{12}$R$_8$ with R=C$_2$H$_5$ [1], R=C$_3$H$_7$ (n-propyl) [2], and R=OSi(CH$_3$)$_2$H [3]. Recently,[4] we applied dynamical decoupling methods in order to suppress nuclear spin diffusion in H@$h_{72}$Q$_8$M$_8$, the derivative with R=OSi(CH$_3$)$_3$. Herein we examine for the first time the effect of deuterium isotopic substitution on the spin relaxation properties of H@$h_{72}$Q$_8$M$_8$, by applying pulsed electron paramagnetic resonance (EPR) methods on its deuterated analogues H@$d_{72}$Q$_8$M$_8$ and D@$d_{72}$Q$_8$M$_8$. For the latter species we measure a phase memory time of 60 $\mu$s at 180 K, the largest obtained so far for this family of molecular spins. We show that selective substitution of encapsulated or peripheral hydrogen atoms with deuterium reveals high-temperature relaxation mechanisms that were previously hidden by proton nuclear spin diffusion. Unusually short $T_M$ values observed for all deuterated species even at liquid helium temperatures are discussed in terms of tunneling reorientation of methyl groups.

[1] Weiden et al, Appl. Magn. Reson. 2001, 21, 507–516.
[2] Schoenfeld et al, Phys. Status Solidi B 2006, 243, 3008–3012.
[3] G. Mitrikas, Phys. Chem. Chem. Phys., 2012, 14, 3782–3790.
[4] G. Mitrikas et al, Phys. Chem. Chem. Phys., 2014, 16, 2378–2383.

Speaker: Dr George Mitrikas (Institute of Nanoscience and Nanotechnology, NCSR Demokritos)
• 16:50
Coherent control of solid state nuclear spin nano-ensembles 25m

Nitrogen-vacancy color centers (NVs) in diamond can be measured at the single cite level even at room temperature, allowing to perform a variety of fundamental experiments.
Here the recent progress in controlling small nuclear spin ensembles at ambient conditions will be presented. A Dynamic Nuclear spin Polarization (DNP) method was developed to transfer the NV’s high (> 92 %) electron spin polarization induced by short laser pulses to the surrounding 13C carbon nuclear spins (I = 1/2). Here the NV is repeatedly repolarized optically, thus providing an effectively infinite polarization reservoir. The polarisation of the nuclear spin ensemble was so high, that it lead to narrowing of the line width of a single NV electron spin transition by a factor of eight [1]. The same technique was used to polarize a macroscopic ensemble of 13C spins, where we achieved an increase of the NMR signal by a factor of 45 [2]. A novel method for both polarization of the nuclear spin bath and its quantitative measurement will be demonstrated - Polarization Read Out by Polarization Inversion (PROPI) [3]. With this technique we are able to determine the exact number of spin quanta transferred from the NV’s electron spin to the nuclear spin bath. Finally, NMR spectroscopy of few tens of nuclear spins is demonstrated by using radio frequency pulses for coherent control and PROPI for polarization and readout of the spin ensemble [4].

References:
[1] P. London at al., Phys. Rev. Lett. 111, 067601 (2013).
[2] J. Scheuer et al., New J. Phys. 18, 013040 (2016).
[3] J. Scheuer et al., Phys. Rev. B 96, 174436 (2017).
[4] T. Unden et al., npj Quantum Information 4, 39 (2018).

Speaker: Dr Boris Naydenov (Institute for Nanospectroscopy, Helmholty Zentrum Berlin für Materialien und Energie, Kekulestr. 5, 12489 Berlin)
• 17:15
Stable radicals tethered to pentacene studied using time resolved EPR and transient absorption spectroscopy 25m

The ability to generate well-defined states with large electron spin polarization is useful for applications in molecular spintronics, high-energy physics and magnetic resonance spectroscopy. Pentacene-radical derivatives can rapidly form triplet excited states through enhanced intersystem crossing and under the right conditions this can in turn lead to polarization of the tethered radical [1]. The magnitude of the spin polarization on the radical substituent depends on many factors: local magnetic and electric fields, molecular geometry, and spin-spin coupling [2-4]. In this work we present time resolved electron paramagnetic resonance (TREPR) and field swept echo detected electron paramagnetic resonance (FSEPR) measurements on three pentacene derivatives with trityl, BDPA or TEMPO substituents. We observe polarization transfer between the pentacene excited triplet and the TRITYL radical, but do not observe the same for the BDPA and TEMPO derivatives. We also investigate polarization transfer in the pentacene-TRITYL system in different glassy environments and observe distinct polarization transfer behavior depending on the solvent used. We explain the TREPR and FSEPR measurements by comparing the excited-state dynamics of the three pentacene derivatives from nanosecond and femtosecond transient absorption measurements. We observe a two order of magnitude difference in the timescale of triplet formation of the pentacene TRITYL system when compared to the pentacene with the BDPA and TEMPO substituents.
1. Chernick, E. T.; Casillas, R.; Zirzlmeier, J.; Gardner, D. M.; Gruber, M.; Kropp, H.; Meyer, K.; Wasielewski, M. R.; Guldi, D. M.; Tykwinski, R. R., J Am Chem Soc 2015, 137, 857-863.
2. Ito, A.; Shimizu, A.; Kishida, N.; Kawanaka, Y.; Kosumi, D.; Hashimoto, H.; Teki, Y., Angew Chem Int Edit 2014, 53, 6715-6719.
3. Jenks, W. S.; Turro, N. J., J Am Chem Soc 1990, 112, 9009-9011.
4. Ishii, K.; Takeuchi, S.; Kobayashi, N. J Phys Chem A 2001, 105, 6794-6799.

Speaker: Dr Claudia E. Avalos (Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne)
• 17:40 18:30
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 17:40
Summarizing the static DNP mechanisms 40m

During the last couple of years, we have been trying to understand the various stages of the DNP process leading to nuclear signal enhancements in static solid solutions, containing free organic radicals. Despite the fact that almost everything is known already, we have made an effort to clarify the basic spin dynamics resulting in these enhancements. For these studies, we performed experiments on the electrons and the nuclei in a variety of amorphous solids. In particular, the combination of EPR and ELDOR spectroscopy together with NMR measurements on the same sample, has led us to formulate computational models for explaining the line shapes of EPR and DNP spectra during microwave (MW) irradiation.
In this presentation, our findings in static samples will be summarized, by comparing simulated spectra of small model spin system with experimentally obtained spectra. For our computations, we use a cross relaxation mechanism to describe electron depolarization and rely on the indirect Cross Effect (iCE) for deriving DNP spectra from EPR spectra. The interaction regimes for the appearance of the Thermal Mixing (TM) phenomena will of course be discussed as well. For the analysis of experimental results, we show how we derive the change of the EPR lineshapes following microwave (MW) irradiation from ELDOR spectra, using the electron spectral diffusion (eSD) and TM models, and again rely on iCE to interpret experimental DNP spectra. Experimental results corresponding to the TM process will also be discussed and the dependence of the enhancements as a function of MW power, MW modulation and radical concentration will be verified.

Note: All static DNP studies have been performed in full collaboration with Daniella Goldfarb and Akiva Feintuch, and our post-doctoral and graduate students.

Speaker: Prof. Shimon Vega (Weizmann Institute of Science)
• 18:30 21:00
Bruker Night 2h 30m Harnack House

Harnack House

• Tuesday, 27 August
• 08:40 10:00
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 08:40
Magnetic resonance for Cellular Structural Biology: from protein structures to functional processes in a cellular context 40m

Magnetic resonance spectroscopies can provide unique contributions to describe cellular processes. Magnetic resonance is indeed suitable not only for characterizing the structural and dynamical properties of biomolecules but, even more importantly, for describing transient interactions and functional events with atomic resolution possibly in a cellular context. This requires the development of suitable methodologies capable of addressing multiple, specific, and sometimes non conventional aspects for describing functional processes in cells.
I will present some examples on how NMR, also integrated with other techniques, can contribute to advance the knowledge on functional cellular processes. They often involve transient interactions suitably studied by NMR, which can also characterize processes in living cells with atomic resolution. Transient interactions occur in metal transfer processes1. The presence of paramagnetic centers, such as iron-sulfur clusters, dramatically affects the NMR spectra, requiring the development of tailored experiments and the integration with multiple techniques, with EPR being essential for these systems. The power of NMR in describing cellular pathways will be presented for processes responsible for copper trafficking and for the biogenesis of iron-sulfur proteins. New major advancements in in-cell NMR2,3 and in the characterization of highly paramagnetic systems4 will be also discussed within an integrated approach where, from single structures to protein complexes, the processes are described in their cellular context within a molecular perspective.

1 Banci L, et al. Affinity gradients drive copper to cellular destinations. Nature 465: 645-648, 2010
2 Banci, L., et al. Atomic-resolution monitoring of protein maturation in live human cells. Nat.Chem.Biol. 9, 297-299, 2013.
3 Luchinat E, Banci L. In-Cell NMR in Human Cells: Direct Protein Expression Allows Structural Studies of Protein Folding and Maturation. Acc Chem Res. 51, 1550-1557, 2018.
4 Banci L, et al. The NMR contribution to protein-protein networking in Fe-S protein maturation. J Biol Inorg Chem. 23, 665-685, 2018

Speaker: Prof. Lucia Banci (CERM and Dept. of Chemistry, University of Florence)
• 09:20
Diffusion and electrophoretic NMR to characterize ion transport in electrolytes 40m

For application of electrolyte materials in energy storage devices their transport properties are essential. Multinuclear (e.g. 1H, 7Li, 19F) Pulsed-Field-Gradient (PFG)-NMR diffusion has become a widely used method in this field. However, to identify the conductivity contribution of specific ion species remains a challenge, since the electrophoretic mobility µ has to be known.
Electrophoretic NMR (eNMR) allows to directly measure the electrophoretic mobility of ions with NMR-active nuclei. During a PFG-NMR experiment an electric voltage is applied and the ion mobility is obtained from its drift velocity in the electric field. Provided that challenges arising from high conductivities and subsequent resistive heating can be overcome, even concentrated electrolyte systems can be investigated.
The lecture reviews our multinuclear eNMR studies on electrolytes for Li battery applications, e.g. Li salt-in-Ionic Liquid systems. Surprisingly, a negative mobility of Li+ may occur, implying a drift direction opposite to the expectation for a cation. This was attributed to a vehicular transport mechanism of Li in net negatively charged anion clusters and has strong implications for battery operation. In some systems a transition from a vehicular to a structural transport mechanism can be achieved by compositional variation.
We further report on various liquid electrolyte systems with organic additives and glyme-based solvate ionic liquids. Here, in addition to ion drift velocities, even a drift of uncharged molecular components in the electric field can be identified by 1H eNMR. Thus, conclusions on their coordination to Li+ ions are possible, sheding light on the mechanisms governing the Li transport.
In summary, electrophoretic NMR elucidates transport mechanisms on a molecular level, and provides unique information; in particular, where correlated motion of different ion species is involved.

Speaker: Prof. Monika Schönhoff (Institute of Physical Chemistry, University of Muenster)
• 10:00 10:30
Coffee 30m
• 10:30 13:00
Biomolecules: Session 14 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 10:30
NMR investigation of the activation mechanism of the guardian of the germ cell TAp63a 35m

Cell fate decisions like cell division or apoptosis require cells to translate signals into a final yes/no answer. Primary oocytes are a special type of cells that are arrested in prophase of meiosis I in which they last for up to 50 years in humans. The number of primary oocytes determines the reproductive capacity of females. Due to the importance and the long arrest time of these cells they have evolved a special type of genetic quality control not present in somatic cells. Regulating this control mechanism is of very high importance: Tight genetic quality control is necessary to maintain the genetic integrity of the entire species but a too stringent mechanism can deplete the whole primary oocyte pool leading to infertility. In female germs cells this genetic quality is monitored by the p53 homolog TAp63α. After DNA damage it gets activated by phosphorylation triggering the transition from a closed dimeric state to an open tetramer. We have used NMR spectroscopy to investigate how phosphorylation determines the critical threshold level for elimination of a primary oocyte. Through measuring single site phosphorylation kinetics in isolated peptides as well as in full-length protein we show that phosphorylation follows a biphasic behavior. We reveal the structural mechanism and show by quantitative simulation that the slow phase determines the threshold of DNA damage that is necessary to induce apoptosis.

Speaker: Prof. Volker Dötsch (Goethe University)
• 11:05
Flavivirus capsid assembly and dynamics: evidence of a structure-driven regulation of protein interaction with intracellular hydrophobic interfaces. 25m

Dengue (DENV) and Zika (ZIKV) are major arthropod-borne human viral disease, for which no specific treatment is available. They are a worldwide important health concern, which causes neurological disorders and hemorrhagic syndrome. Although the structure of ZIKV and DENV virion has been determined, information on the nucleocapsid is lacking. The most accepted hypothesis is of a disorganized nucleocapsid. Using NMR, we solved the structure and dynamics of full length ZIKV capsid protein (ZIKVC) and the dynamics of DENV capsid protein (DENVC). We showed that the addition of oligonucleotides can form an organized nucleocapsid-like particles (NC-like). The binding to intracellular hydrophobic interfaces, such as endoplasmic reticulum and/or lipid droplets is essential for virus replication. The hydrophobic cleft is the binding site, along with the intrinsically disordered region, and an open-close dynamic of the globular domain that are species-specific. For ZIKVC, α-helix 1 is smaller and partially occludes protein hydrophobic cleft. Measurements of the dynamics of α-helix 1, surface exposure and thermal susceptibility of each backbone amide hydrogen in protein structure revealed the occlusion of the hydrophobic cleft by α1/α1´ and supported a α-helix 1 position uncertainty. Based on the findings, we propose that the dynamics of flaviviruses structural elements responds for a structure-driven regulation of protein interaction with intracellular hydrophobic interfaces, which would impact in the switches necessary for nucleocapsid assembly. Subtle differences in the sequence of helix 1 impact on its size and orientation and on the degree of exposure of the hydrophobic cleft, suggesting that α-helix 1 is a hotspot for evolutionary adaptation of flaviviruses’ capsid proteins.
Acknowledgements: FAPERJ, CAPES, CNPq, INBEB-CNPq.

Speaker: Prof. Fabio C. L. Almeida (Federal University of Rio de Janeiro (UFRJ))
• 11:30
Progress of the structural characterization on a eukaryotic rhodopsin by solid-state NMR 25m

Magic-angle-spinning solid-state NMR (MAS-SSNMR) has emerged as a powerful technique of structural biology. It is particularly attractive for its unique capability of providing structure and dynamic for membrane proteins in lipid bilayers. In this presentation I will introduce our recent progress in structure and dynamic characterization of Leptosphaeria rhodopsin (LR).
LR was the first discovered eukaryotic light-driven proton pump, which uses light energy to transport protons across the cell membranes. LR shares the typical heptahelical topology and has a retinal covalently bound to the protein core. However, the structure and the detailed mechanisms of LR are still unknown. The proteins were prepared by P. pastoris expression system. To determine the structure of LR, the 2D and 3D MAS-SSNMR spectra were collected to achieve backbone and side-chain assignments. Sparsely 13C labeled protocol for P. pastoris expression systems were developed to obtain long-range distances for structural illustrations. The LR forms homo-trimers in lipid environments. Paramagnetic relaxation enhancements were applied in characterization of the intermonomer interface.
The LR shares heptahelical transmembrane topology. The hydrogen-bonding networks formed by the proton of the protonated Schiff-base and critical Asp residues are the key elements for LR function. The chemical shift of Asp residues suggested the carboxyl sidechains of D139 and D266 are deprotonated, consistent with the common knowledge of microbial rhodopsin. On the other hand, the solid-state H/D exchange experiments suggested the rapid exchange between solvent water and the proton of the protonated Schiff-base. This indicated the participation of water molecule in the pathway of proton pump of LR.

Speaker: Prof. Shenlin Wang (Peking University)
• 11:55
Insights into the Antifungal Activity of Amphotericin B from Solid-State NMR 35m

In this talk, I will describe ongoing efforts in my laboratory (in collaboration with Prof. Martin D. Burke at Illinois) to understand the mode of action of the gold standard antifungal drug amphotericin B (AmB). We have previously proposed a hypothesis that AmB acts as a sterol sponge, a high molecular weight assembly that cooperatively assembles and extracts ergosterol from the yeast plasma membrane. Binding of ergosterol is correlated with antifungal activity and binding of cholesterol with toxicity. Thus the sponge model predicts that analogs of AmB with greater binding specificity for ergosterol v. cholesterol will have an improved therapeutic index. The structural basis for this activity, however, remains incompletely understood. We have developed and implemented experiments involving 13C-labeled AmB both alone and in complex with sterols, in order to understand the detailed conformational rearrangements and structural motifs that endow AmB with these unique biophysical properties.

Speaker: Prof. Chad M. Rienstra (University of Illinois at Urbana-Champaign)
• 12:30
Assessing site-specific water accessibility in folded and unfolded proteins using hyperpolarization-enhanced 2D HMQC NMR 30m

Hyperpolarized water is a valuable aid in biomolecular NMR. One can utilize it to achieve, under physiologically-like conditions, amide group polarizations that are orders-of-magnitude larger than their thermal counterparts. Suitable experimental procedures can exploit this to deliver 2D 1H-15N NMR correlations, with good resolution and enhanced sensitivity. The resulting signal enhancements depend on the exchange rates between amides and water protons, yielding information about solvent accessibility. This study applied the ensuing "HyperW" NMR method to four proteins, which exhibit a gamut of exchange behaviors. These included PhoA(350-471), an unfolded fragment of Alkaline Phosphatase from E. coli; barstar, a folded ribonuclease inhibitor from Bacillus amyloliquefaciens; R17, a system possessing folded and unfolded forms under slow interconversion; and drkN-SH3, an N-terminal protein domain where folded and unfolded forms interchange more rapidly and with temperature-dependent population ratios. For the unstructured PhoA4 fragment 2D HyperW sensitivity enhancements were very high, ≥300× over their thermal counterparts, expected due to fast amide exchanges that occur throughout this unfolded protein sequence. Though fully folded barstar also exhibited substantially-enhanced residues; these were not uniform, and reflected what appeared well folded but surface exposed residues. R17 showed the expected superposition of ≥100-fold enhancements for its unfolded form, coexisting with more modest enhancement for the folded. The behavior of drkN-SH3 domain was unexpected: HyperW substantially enhanced both folded and unfolded states -but foremost of all certain sites of the folded protein. A number of explanations– including cross-correlated relaxation processes, and the possibility of three-site exchange magnetization transfers– were considered to account for these preferential enhancements. Still, the most “reasonable” explanation for larger folded-site enhancements, appears to be that faster exchange rates characterize these sites than their unfolded counterparts. We discuss factors that could bring about such anomalous, hitherto unobserved behavior departing from accepted paradigms relating solvent exposure and protein fold.

Speaker: Prof. Lucio Frydman (Department of Chemical and Biological Physics, Weizmann Institute of Science)
• 10:30 13:00
Computation: Session 11 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 10:30
Splitting hairs: Small physical effects in NMR 35m

Computation of conventional NMR parameters has become routine. There remain, however, interesting physics to be explored. I introduce four topics of this kind.
For the first time, measured $J$-coupling over a van der Waals (vdW) bond, between 129Xe and 3He in a gas-phase nuclear spin co-magnetometer, can be directly compared with quantitative calculations [1]. The latter involve the second virial coefficient of $J$(129Xe-3He) from relativistic potential energy and $J$-coupling curves.
NMR parameters are magnetic field-dependent. So far, only $B$0-dependent 131Xe quadrupole coupling has been found [2]. Experiments on Co(acac)3 -complex [3], where low-lying d-d excitations render the 59Co shift significantly $B$0-dependent, are compared with non-linear response theory for the leading $O$($B$02) terms [4].
Nucleus-specific information can be obtained from changes in light beam polarisation due to interaction with nuclear spins [5]. We predict nuclear spin-induced magnetochiral birefringence and dichroism, as possible reporters of chirality via different indices of refraction when light propagates parallel and antiparallel with spin magnetisation [6].
In a multi-scale study of spin-exchange optical pumping [7], Rb-Xe collisions are extracted from molecular dynamics and analysed using spin dynamics driven by a quantum-chemical spin Hamiltonian. We reproduce the roles of binary collisions and long-lived vdW complexes and, for the first time, see step-wise build-up of 129Xe polarisation upon vdW oscillations.
[1] J. Vaara, M. V. Romalis, Phys. Rev. A, in press (2019)
[2] T. Meersmann, M. Haake, Phys. Rev. Lett. 81, 1211 (1998)
[3] A. M. Kantola, P. Lantto, I. Heinmaa, J. Vaara, J. Jokisaari, in preparation (2019)
[4] P. Manninen, J. Vaara, Phys. Rev. A 69, 022503 (2004)
[5] I. M. Savukov, S.-K. Lee, and M. V. Romalis, Nature 442, 1021 (2006)
[6] L.-j. Fu, J. Cukras, D. Fedotov, S. Coriani, J. Vaara, in preparation (2019)
[7] J. Rantaharju, M. Hanni, J. Vaara, submitted (2019)

Speaker: Prof. Juha Vaara (University of Oulu)
• 11:05
Second order dispersion by optimised rotation pulses 25m

The GRAPE method of optimal control can attempt to find the maximum overlap between a desired rotation propagator and the effective propagator of the pulse sequence [1], termed the fidelity. In finding optimal rotation pulses, numerical optimisation methods use the gradient of the fidelity to give super-linear convergence to a maximum overlap [2].

Building on past research that creates broadband pulses performing unitary propagators (BURBOP) [3,4], the research presented in this communication advances a step forward to create a new class of pulses with a defined second order phase dispersion.

One of the problems associated with universal rotation solutions, named BURBOP pulses [3,4], is the resulting high irradiation energy compared with the easier control problem of optimising state-to-state problems. A novel method is presented which will show this energy can be lowered by defining target rotation propagators as a function of phase dispersion. A customised version of the Spinach [5] optimal control toolbox [2,6,7] is used to simulate an ensemble of two-level quantum systems. This new class of pulse is named SORDOR pulses by the authors.

This optimal control method uses a defined quadratic phase dispersion, similar to the chirped pulses, for the targets of optimal control methods to find pulses that produce a rotation around an axis [3,4] at each frequency offset. Results for $90^{\circ}$ and $180^{\circ}$ SORDOR pulses are compared the achievable fidelity to the equivalent BURBOP pulse.

[1] J Magn. Reson., 172, 296 (2005)
[2] J Magn. Reson., 212, 412 (2011)
[3] J. Magn. Reson. 225, 142 (2012)
[4] J. Magn. Reson. 216, 78 (2012)
[5] J. Magn. Reson., 208, 179 (2011)
[6] J. Chem. Phys., 143, 084113 (2015)
[7] J. Chem. Phys., 144, 204107 (2016)

Speaker: Dr David Goodwin (Karlsruhe Institute of Technology)
• 11:30
First-principles computations of NMR shifts for extended paramagnetic solids: significant effects beyond the contact shifts 25m

NMR is a powerful tool for studying the structural and electronic properties of paramagnetic solids. However, the interpretation of paramagnetic NMR spectra is often challenging as a result of the interactions of unpaired electrons with the nuclear spins of interest. Recently, we reported a novel protocol to compute and analyze NMR chemical shifts for extended paramagnetic solids, accounting comprehensively for Fermi-contact (FC), pseudo-contact (PC), and orbital shifts.[1,2] We combine periodic DFT computation of hyperfine and orbital-shielding tensors with an incremental cluster model for g- and zero-field-splitting (ZFS) D-tensors. The hyperfine tensors are computed with hybrid DFT functionals using the highly efficient Gaussian-augmented plane-wave implementation of the CP2K code. The incremental cluster model allows the computation of g- and ZFS D-tensors by ab initio complete active space self-consistent field and N-electron valence-state perturbation theory methods. We find that 7Li shifts in the high-voltage cathode material LiCoPO4 are dominated by spin-orbit-induced PC contributions, in contrast to previous assumptions, changing the interpretation of the shifts fundamentally in terms of covalency. Similar protocols can be applied to the computation of pNMR shifts for clusters with multiple paramagnetic centers. Using such a procedure, 1H and 13C shifts have been computed for derivatives of the porous Cr-MIL-101 solid, which contain Cr3O clusters with magnetically coupled metal centers within the metal-organic frameworks.[3] A combination of experimental and computational methods has been used to explore the competitive small-ligand binding to these MOFs. The developments described pave the way towards a more-widespread computational treatment of NMR shifts for paramagnetic materials.[4]

[1] Mondal, A.; Kaupp, M. J. Phys. Chem. Lett., 2018, 9, 1480-1484.
[2] Mondal, A.; Kaupp, M. J. Phys. Chem. C, 2019, 123, 8387-8405.
[3] Wittmann, T.; Mondal,A.; et al. J. Am. Chem. Soc., 2018, 140, 2135-2144.
[4] Mondal, A.; Kaupp, M. Solid State Nucl. Magn. Reson.,2019 (in press)

Speaker: Dr Arobendo Mondal (Technical University of Munich, Germany)
• 11:55
Computational methods for NMR crystallography of zeolites 35m

Solid-state nuclear magnetic resonance (SSNMR) spectroscopy has emerged as an important technique for structural characterization of solids. Due to the fact that it provides local structural information about the environments of NMR-active nuclei, SSNMR is highly complementary to diffraction techniques whose strength lies in providing information about the long-range periodic structure of a material. By combining solid-state NMR and diffraction techniques with various computational methods (modeling, density functional theory, etc), powerful approaches to structure determination of materials are being developed. These integrated structure determination strategies in which SSNMR spectroscopy plays a crucial role is broadly referred to as NMR crystallography. This talk will provide an overview of our NMR crystallography strategies for solving and refining zeolite crystal structures using advanced 29Si SSNMR methods and employing insights from graph theory, tiling theory, and machine learning.

Speaker: Prof. Darren Brouwer (Redeemer University College)
• 12:30
Methionine renaissance: computing methyl NMR assignments from X-ray structures 30m

Methyl groups are privileged probes for the NMR study of large proteins. Methionine is the least abundant of the methyl-containing amino acids but is often directly connected to functionally important sites. While isotopic labeling with 13CH3-methionine is straightforward, assignment of the resulting spectra is challenging and often requires site-specific mutation of each of the methionines in the protein. Standard methods based on through-bond correlation to assigned backbone resonances or by matching observed and predicted inter-methyl NOEs on the basis of known X-ray structures are not applicable to exclusively methyl-methionine labeled proteins.
In this communication we will show that methionine methyl groups chemical shifts can be predicted from X-ray structures based on fast quantum chemical calculations on simplified models. The use of quantum chemical calculations outperforms the statistics-based predictions in the case of the scarce and flexible methionine residues. The calculated chemical shifts successfully predict the correct assignment or, at least, helps to define the minimal number of mutations needed to complete it.
Methyl methionine NMR has been used to functionally characterize the catalytic domains of calcineurin (345 residues, 7 methionines) and Src (253 residues, 10 methionines).
In the case of calcineurin, methionine methyl signals probe the binding of peptides and the conservation of a cis peptide bond in the mutant in which the native P84 was replaced by alanine [1].

Acknowledgements. Supported in part by grant BIO2016-78006R and by access to the R-LRB, the Spanish NMR facility network.

Reference

1. Teixeira, J.M.C., Guasch, A., Biçer, A., Aranguren-Ibáñez, A., Chasmniam, S., Paniagua, J.C., Pérez-Riba, M., Fita, I. , Pons, M. Cis-trans proline isomers in the catalytic domain of calcineurin. FEBS J. 286, 1230-1239 (2019)
Speaker: Prof. Miquel Pons (University of Barcelona)
• 10:30 13:00
Dynamics: Session 13 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 10:30
Allostery and Dynamics in Ion Channels and Oligomeric Proteins 35m

Allostery in ion channels controls activation coupled inactivation and partly controls mean open time. Solid state NMR experiments on full length wild type channel in proteoliposomes provide evidence for evacuation of ions from the selectivity filter during inactivation and strong coupling between opening and ion affinity. Furthermore, a number of site specific mutants altered in their inactivation properties in the hinge of the inner helix (e.g. F103A) suggest that a group of bulky residues serve as “hotspots” for allostery. The plasticity of ion channels is clearly critical to the many essential processes they carry out in all cells. Methods for characterizing millisecond and microsecond conformational exchange processes in high resolution SSNMR experiments will also be discussed.

The talk will also discuss structures of amyloids involved in human biology, and new NMR methods to sensitize detection of signals. RIPK1:RIPK3 core complex of the necrosome, which initiates TNF-induced necroptosis in the context of immune defense, cancer and neurodegenerative diseases. Using solid-state NMR, we determined the high-resolution structure of the core. RIPK1 and RIPK3 assume serpentine conformations, with short β-segments. Packing analogous to other amyloids results in a hydrophobic core with both hetero and homo hydrophobic contacts, and unusual exposed “ladders” of interacting amino acids. The molecularly detailed structure of a hetero-oligomeric amyloid and provides insights into the mechanisms of signal transduction and of inhibition of necroptosis.

Speaker: Prof. Ann McDermott (Columbia University)
• 11:05
A high-resolution description of functional dynamics and allosteric coupling of the β1-adrenergic receptor from backbone NMR 25m

G protein-coupled receptors (GPCRs) are physiologically important transmembrane signaling proteins that elicit intracellular responses upon binding of ligands on the extracellular site. Breakthroughs in crystallography have provided a wealth of static GPCR structures ranging from ligand-bound inactive receptors to fully active receptors in complex with intracellular binding partners such as heterotrimeric G protein and its mimetics. However, dynamical information on the different functional receptor states and their transitions is scarce. Such information is needed to understand the mechanisms of receptor regulation and signal transmission.
We have previously shown that the GPCR response to various ligands can be followed from 1H- 15N resonances at virtually any backbone site in a thermostabilized mutant of the turkey β 1-adrenergic receptor (β 1AR) [1]. We now provide a detailed analysis of populations and dynamics derived from 15N chemical shifts and relaxation rates. For this we used the fully thermostabilized and a more native-like mutant of the receptor in binary complexes ranging from antagonists to agonists as well as in the ternary agonist●G protein mimetic complex. This provides new insights into its activation mechanism and key residues involved in allosteric signal transmission.

[1] Isogai, S., Deupi, X., Opitz, C., Heydenreich, F. M., Tsai, C.-J., Brueckner, F., Schertler, G. F. X., Veprintsev, D. B., Grzesiek, S., Nature 2016, 530, 237–241.

Speaker: Dr Anne Grahl (Biozentrum, University of Basel, Switzerland)
• 11:30
13C-detected NMR methods to characterise side-chain behaviour in large molecular systems 25m

The behaviour of side chains is fundamental to the biology and pathology of proteins. They play essential roles in processes as diverse as folding, catalysis, binding and allosteric regulation, and it is clear that in many cases their function is as much linked to their dynamic behaviour as their structure. Despite their significance, methods probing the behaviour of side chains are limited, and mainly focus on small proteins and residues containing methyl groups.

13C-detected NMR spectroscopy in a per-deuterated environment provides an excellent means of probing residues and systems intractable to conventional 1H-detected methods. The presented suite of pulse sequences[1] make use of a single, uniformly-labelled sample to characterise a range of non-methyl- and methyl-bearing side chains. The base residue-specific carbon-carbon correlation experiment has been extended to include elements reporting on both the structure and dynamic behaviour of these side chains. Chemical exchange saturation transfer (CEST) experiments report on conformational exchange on a millisecond time-scale, whilst long-range 13C-13C scalar couplings report on nanosecond to millisecond motions. Most importantly, these experiments have been used to investigate systems as large as 82 kDa. The presented class of methods promises characterisation of side-chain behaviour in large systems and at a level that has so far been reserved for the protein backbone.

[1] Pritchard & Hansen 2019 Nature Communications 10:1747

Speaker: Dr Ruth B. Pritchard (University of Sussex)
• 11:55
Inspection of solution-state NMR data to evaluate protein conformational changes 35m

NMR studies of large proteins, over 100 kDa, in solution are technically challenging and thereby of considerable interest in the NMR field. This is primarily due to slowing of molecular tumbling in solution as molecular mass increases. Typical 1H-13C or 1H-15N correlation spectra using 13C- or 15N uniformly labeled proteins show severe line-broadening and signal overlap. It is well known that selective isotope labeling, often concomitant with deuteration, is a useful strategy to reduce signal overlap and line-broadening in biomolecular NMR. However, a reduction in the number of signals is, in turn, disadvantageous in characterization of the overall protein feature. Thus, inspection of solution state NMR data not only of 1H-13C correlation spectra recorded using the selectively-labeled proteins, but also of 1H-15N correlation spectra of uniformly 15N-labeled protein is still useful. We discuss consistency in NMR data recorded using different NMR nuclei for a 66 kDa protein, HIV-1 reverse transcriptase precursor, that forms a homodimer with micro-molar dissociation constant in solution, to understand the structural characteristics. This work is supported by NIH NIGMS and NIAID (GM105401 and GM082251).

Speaker: Dr Rieko Ishima (University of Pittsburgh School of Medicine)
• 12:30
Lanthanide-induced relaxation anisotropy 30m

Lanthanide ions accelerate nuclear spin relaxation by two primary mechanisms: dipolar and Curie. Both are commonly assumed to depend on the length of the lanthanide-nucleus vector, but not on its direction. In this communication, we demonstrate experimentally and verify theoretically that this is wrong – careful proton relaxation data analysis in a series of isostructural lanthanide complexes (Ln=Tb, Dy, Ho, Er, Tm, Yb) reveals angular dependence in both Curie and dipolar relaxation. The reasons are:

1. that magnetic susceptibility anisotropy can be of the same order
of magnitude as the isotropic part (contradicting the unstated
assumption in Gueron‘s theory of the Curie relaxation process [1]);

2. that zero-field splitting can be much stronger than the electron
Zeeman interaction (Bloembergen’s original theory of the
lanthanide-induced dipolar relaxation process makes the opposite
assumption [2]).

These factors go beyond cross-correlation effects; they alter the relaxation theory treatment and make angular dependencies appear in the nuclear spin relaxation rates. Those angular dependencies are impossible to ignore – we demonstrate this both theoretically and experimentally, and suggest that a major revision is needed of the way lanthanide-induced relaxation data is used in structural biology.

Speaker: Dr Elizaveta Suturina (University of Bath)
• 10:30 13:00
Hyperpolarization: Techniques: Session 12 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 10:30
Confining and Quantifying Hyperpolarization: 460 GHz-700 MHz DNP NMR using Closed-Cycle Helium MAS and Dual Gyrotron Setup 35m

Sensitivity of magic-angle spinning (MAS) solid-state NMR has been dramatically improved by the advent of high-field dynamic nuclear polarization (DNP) techniques through numerous discussions and breakthroughs made for improving the signal enhancement factor. Beyond the discussions on the sensitivity gain, we here propose two new methods to pursuit hitherto under-explored curiosity: a method to confine the hyperpolarization for spatially selective observation of mesoscale molecular domains, and a method that enables quantitation of absolute 1H polarization amplitude and its spatial distribution around a radical molecule (polarizing agent). The former method utilizes our unique double-gyrotron setup and its ability to switch the microwave frequency back and forth over the range of 0.7 GHz in synchrony to the RF pulses. Each microwave frequency is set to excite the positive or negative DNP effect in turn, producing a sort of “polarization wave” in space around the radical molecule. The second method is based on the use of a closed-cycle helium MAS system for ultra-low temperature DNP, enabling the total sensitivity gain exceeding a factor of 1000 at T = 30 K and B0 = 16.4 T. In such a case, the high-order spin-correlated term (2 IzSz) in the quasi-equilibrium spin density operator grows in a significant amplitude and, as we show, is observable separately from the lowest-order Zeeman term (Sz) for the polarization quantitation. The method does not require evaluation of “microwave-off” signal as well as un-doped reference sample, and is also unaffected by the quenching and depolarization effects, providing an accurate and efficient way for the polarization quantitation. Potential applications will also be discussed.

Speaker: Dr Yoh Matsuki (Osaka University)
• 11:05
Multi-Sample Dissolution DNP with a Cryogen-Free Polariser 25m

Dissolution Dynamic Nuclear Polarisation (DNP) has shown great potential in providing large signal enhancement to metabolites of interest in low gamma metabolic magnetic resonance imaging. Originally DNP polarisers were based on pumped-helium cryostats, which provide a high cooling power to contain the extra heat load introduced during the dissolution. However, these systems are not efficient at running at low temperatures for extended periods of time; neither are they cost effective due to the rising price of helium.

We present a closed-cycle cryogen-free 7T polariser which requires no input of liquid helium or any other cryogens. The polariser is based on a modified commercial dilution refrigerator. The closed system can run continuously at 1.4K for many weeks without interruption, useful for solid state measurements of samples with prolonged longitudinal relaxation times as well as making it a highly interesting system for dissolution DNP. Liquid-state 13C polarization larger than 40% were obtained on different samples.

Traditional methods of sample dissolution are not suitable for a cryogen-free system due to the need to introduce warm helium to pressurise the sample space prior to introduce the dissolution apparatus into the cryostat. Instead, in order to minimise the heat load introduced during the dissolution process, we used a fluid path.

The sample space in the polariser is sufficiently large to house a maximum of 4 fluid paths meaning up to 4 mL of sample can be polarised at once. We implemented an insert that allows polarising two samples in parallel and dissolving them consecutively within an interval of 20min. These consecutive dissolutions can be carried out without significant deleterious heat-loads at the cooling stages of the cryostat. Limiting temperature increases on any of the cooling stages in this way allows a rapid recovery of the base temperature and prevents a potential quench of the magnet.

Speaker: Dr Adam Gaunt (University of Cambridge)
• 11:30
Catalyzing the progress in parahydrogen-based NMR hyperpolarization 25m

Among the hyperpolarization (HP) techniques, parahydrogen-based methods are the simplest and technically least demanding. Because such techniques (PHIP, SABRE) rely heavily on catalysis, some of the unsolved problems in both fields are rather similar. One major trend in modern catalysis is a broad search for approaches to combine advantages of homogeneous and heterogeneous catalysts, namely the well-defined structure of the active catalytic center of homogeneous catalysts for high reaction selectivity, and the ease of solid heterogeneous catalyst removal after the reaction. Similarly, it would be highly advantageous to combine the ability of a soluble transition metal complex to add H2 to a substrate in a pairwise manner, and the feasibility of rapid filtration of the reaction mixture to yield a metal-free HP fluid for biomedical use. Indeed, HP effects have been demonstrated lately with the use of several concepts of modern catalysis, including immobilization of transition metal complexes on porous solids, the use of single-site and single-metal-atom heterogeneous catalysts, and active site isolation in metal alloys and bimetallic structures. Heterogeneous catalysis is also suitable for production of HP propane as a promising agent for gas-phase imaging. Another recent breakthrough in modern catalysis is the demonstration that certain metal-free systems are able to activate small molecules such as H2. PHIP effects with the use of amine-borane frustrated Lewis pairs and other metal-free systems demonstrated recently may provide an alternative way to produce biocompatible HP solutions. Thus, the implementation of the achievements of modern catalysis in HP research can lead to a substantial progress in parahydrogen-based NMR signal enhancement, as will be illustrated with several recent examples. Furthermore, the HP techniques can be highly useful in addressing many challenges of modern catalysis research, to be exemplified with the mechanistic insight into the heterogeneous hydrogenation of cyclopropane and MRI of model hydrogenation reactors.

Speaker: Prof. Igor V. Koptyug (International Tomography Center, SB RAS)
• 11:55
Utilizing hyperpolarized noble gas T1 relaxation contrast for MRI in biomedical and engineering applications. 35m

Longitudinal (T1) relaxation is usually considered as disadvantageous for MRI with hyperpolarized (hp) spin systems as it leads to depolarization and hence to a loss in the observable signal. However, it has been demonstrated previously that quadrupolar T1 relaxation of the hyperpolarized noble gas isotope 83Kr (nuclear spin I = 9/2) can utilized to probe surfaces that are in contact with the noble gas. For example, surface quadrupolar relaxation (SQUARE) T1 maps of hp 83Kr are indicative of an emphysema model in excised rodent lungs [1]. MRI at the very low resonance frequency of 83Kr (i.e. 11.5 MHz at 7 T) requires hyperpolarization through spin exchange optical pumping (SEOP) similar to that for the hp 129Xe production. However, as a consequence of quadrupolar relaxation, hp 83Kr cannot be concentrated from buffer gases of the laser pumping process through cryogenic separation or through membranes without depolarization. Therefore, a new production methodology was developed that uses molecular hydrogen as buffer gas during SEOP and its subsequent removal through catalytic combustion [2]. Currently, novel instrumentation is being developed to make this approach feasible for clinical applications.
Similar to 83Kr MRI SQUARE contrast, paramagnetic relaxation of hp 129Xe can be applied to study surfaces, in particular for chemical engineering and materials science applications. Generally, MRI of fluid flow can probe the structure-transport relationship [3], and we use hp 129Xe to study gas transport and reactive zones in diesel catalysts that consist of materials with hierarchical pore structure. The accessibility of catalytic and paramagnetic centers can be probed through 129Xe relaxation measurements provide insights into catalytic activity in these systems.

[1] DML Lilburn et al., J. R. Soc. Interface, 12, (2015), 20150192.
[2] NJ Rogers et al., Proc. Nat. Acad. Sci., 113, (2016), 3146-3168.
[3] GE Pavlovskaya et al, Physical Review Fluids, 3, (2018), 044102_1-20.

Speaker: Prof. Thomas Meersmann (University of Nottingham)
• 12:30
75% Liquid-State 1H Polarization for Hyperpolarized Water 30m

Hyperpolarization via dissolution Dynamic Nuclear Polarization (dDNP) is without doubt the most widespread technique to overcome the low sensitivity in the liquid state Magnetic Resonance.1 Hyperpolarized water is a versatile tool with possible applications ranging from biomedicine to chemistry. For instance, it can be used to acquire high resolution angiographic and perfusion images without employing any metal-ion contrast agent (e.g. Gd3+) in animals,2 or probe proton exchange in proteins.3

Nevertheless, exploiting the 1H nuclei enhanced signal is not as straightforward as for 13C or other low-gamma nuclei: not only is the T1 of pure water intrinsically too short on the dDNP scale (3-4 s),4 but the strong interaction between water protons and paramagnetic agents in solution provides a fatal relaxation rate contribution during dissolution and transfer.

UV-induced non-persistent radicals have been employed to efficiently polarize 13C and other low-gamma nuclei via dDNP.5 Generated by UV-irradiation of a frozen solution containing a fraction of pyruvic acid or its derivatives, these radicals are stable as far as the sample temperature is below 190 K. The UV-radicals natural quenching at the moment of dissolution alleviates from the radical elimination step, and drastically reduces relaxation processes during dissolution and transfer.

Here we show that pyruvic acid derivates UV-irradiated for 10 min at 77 K can generate water solid-state 1H polarizations higher than 90% within 20 min at 6.7 T and 1.1 K, leading to radical-free liquid-state water 1H polarizations of ~ 75 % with liquid-state 1H T1s higher than 40 s at 9.4 T and 313 K.

1 J. H. Ardenkjaer-Larsen et. al, Proc Natl Acad Sci, 2003
2 K. W. Lipsø et. al, Mag Res Med, 2017
3 Q. Chappuis et. al, J Phys Chem Lett, 2015
4 K. Krynicki, Physica, 1966
5 A. Capozzi et. al, Nat Com, 2017

Speaker: Dr Arthur C. Pinon (Technical University of Denmark)
• 10:30 13:00
Materials: Session 15 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 10:30
Defects within Solid Materials Elucidated using NMR Spectroscopy – from Local Vacancies to Mesoscale Disorder 35m

Structure-property relations are essential for designing materials. As many properties are governed by defects and disorder, respectively, elucidating structural details on various length scales is a cornerstone for material science and solid-state chemistry. This lecture will give an overview of our recent progress on using solid-state NMR spectroscopic techniques for studying defects and disorder. Hereby, we will show, that NMR aids the structure elucidation process for materials as diverse as high-pressure minerals, frameworks and supramolecular self-assemblies. It provides central information from local to intermediate length scales by exploiting chemical shifts, connectivities, distances and orientation correlations based on homo- and heteronuclear correlation experiments. NMR spectroscopy is at its best, when used quantitatively as a cost function in addition to scattering and quantum mechanical calculations [1 - 3].
We will report on the incorporation of water in anhydrous ringwoodite by formation of various hydroxyl defects [2, 3]. Here the quantitative analysis of 1H 1D and 2D DQ NMR spectra allowed for unravelling a surprisingly rich defect chemistry. In contrast, the key to a deeper understanding of substitution defects within Bridgmanite was an unambiguous assignment of the 27Al MAS NMR spectra based on STMAS experiments. Finally, to derive a mechanistic picture for the supramolecular self-assembly of benzene trisamides (BTAs) [4] and the role of the resulting nanoobjects within the foaming process of polypropylene, multinuclear (1H, 13C, 15N and 19F) and multidimensional NMR experiments were carried out. DNP was essential to allow for studying the BTA polymer mixtures down to concentrations of a few hundred ppm.

[1] H. Grüninger, et. al. J. Am. Chem. Soc. 2017, 139, 10499.
[2] H. Grüninger, et al. Phys. Chem. Chem. Phys. 2018, 20, 15098.
[3] C. S. Zehe, et al., Angew. Chem. Int. Ed., 2017, 56, 4432.

Speaker: Prof. Juergen Senker (University of Bayreuth)
• 11:05
125Te broadband solid-state NMR of the Dirac edge states in ultrathin Bi$_{2}$Te$_{3}$ nanoplatelets 25m

Detection of metallic Dirac electron states on the surface of topological insulators${^1}$ is, to date, restricted to a small number of experimental techniques, such as angle resolved photoemission spectroscopy and scanning tunneling microscopy. The encroachment of the Dirac states into the bulk interior of a topological insulator is yet to be illuminated experimentally. Getting insight, is crucial in order to further understand the physics of topological materials and probe key properties${^2}$ of this material class. Due to the dependence of the spin orientation of the Dirac electron coupling with the orbital motion and the propagation of this interaction in a crystal, a probe that is sensitive to both spin and orbital motion is necessary. Solid-state Nuclear magnetic resonance (ssNMR) appears to fulfill these requirements, as the nuclear magnetic shielding, and consequently the NMR frequency shift, depends on the spin and orbital magnetic susceptibility at the position of each resonating nucleus. Here, combining advanced, high-resolution broadband$^{3,4}$ solid-state $^{125}$Te NMR methods with state-of-the-art density functional theory calculations and scanning transmission electron microscopy on ultra-thin Bi$_{2}$Te$_{3}$ nanoplatelets, we demonstrate an excellent atomic-scale probe of the Dirac electrons, mainly through the isotropic NMR Knight shift induced by the Dirac electron orbital currents. In this way, the NMR Knight shift and spin relaxation due to the Dirac electrons at all non-equivalent Te positions were acquired, unveiling the way that Dirac electrons and their excitations spread into the interior of the nanoplatelets.

1. M. Z. Hasan, C. L. Kane. Rev. Mod. Phys. 82, 3045-3067 (2010).
2. S. Wu, et al. Science 359, 76–79 (2018).
3. Clement, R. J, et al. J. Am. Chem. Soc. 2012, 134, 17178– 17185.
4. Pell, A. J, et al. State.Prog. Nucl. Magn. Reson. Spectrosc. 111, 1-271, 2019.
Speaker: Mr Wassilios Papawassiliou (Department of Materials and Environmental Chemistry,Stockholm University)
• 11:30
Transport of Organic Electrolytes and Ionic Liquids in Carbon Materials for Supercapacitors: The High-Gradient NMR Approach 25m

The molecular diffusion of ions in energy storage devices, such as, e.g., supercapacitors, is the process enabling their charging and discharging ability. Chmiola et al. demonstrated the strong impact of micropores on the increase of specific capacitance using a series of titanium carbide-derived carbons exhibiting different but precisely uniform pore sizes [1]. An anomalous increase in specific capacitance was observed for those pores being comparable in size to adsorbed electrolyte ions, while the larger pores led to the loss of capacitance. However, under such constraints, one expects kinetic problems caused by confinement-induced obstruction for molecular diffusion [2].
Only very recently, the direct experimental assessment of the ion transport characteristics within the pores of carbon materials became accessible using the quasielastic neutron scattering [3] and the pulsed field gradient (PFG) NMR [4].
Inspired by these recent methodological achievements, we applied the PFG NMR techniques to directly (and selectively) probe the diffusion characteristics of each individual component of organic electrolytes and ionic liquids, that is anion and cation (and solvent, when present), confined to model carbons with uniform and well-defined pore sizes - microporous, mesopores, and with hierarchical pore organization [5]. Quite unexpectedly, it is observed that the presence of a network of mesopores, in addition to smaller micropores—the concept widely used in heterogeneous catalysis to promote diffusion of sorbates—does not necessarily enhance ionic transport in carbon materials.

[1] J. Chmiola et al., Science 313 (2006) 1760‑1763.
[2] A. Lee et al., Nanotechnology 25 (2014) 315401.
[3] N. Osti et al., Phys. Rev. Materials 1 (2017) 035402.
[4] A. Forse et al., Nat. Energy 2 (2017) 16216.
[5] L. Borchard et al., Adv. Energy Mater. 111 (2018) 1800892.

Speaker: Dr Muslim Dvoyashkin (Institute of Chemical Technology, Universität Leipzig)
• 11:55
Solid-state NMR studies of the electrochemical cycling of LiNi$_{0.8}$Mn$_{0.1}$Co$_{0.1}$O$_2$ cathodes 35m

The layered oxide LiNi$_{0.8}$Mn$_{0.1}$Co$_{0.1}$O$_2$ (NMC811) is a promising future cathode material for lithium-ion batteries in electric vehicles due to its high specific energy density. The practical use of NMC811 cathodes, however, faces difficulties as they suffer from fast capacity fade. Mitigating this performance fade requires detailed knowledge of the changes of structure and dynamics of NMC811 during charge and discharge.

$^7$Li solid-state NMR is a well-suited technique for investigating lithium-ion battery materials as it is sensitive to the local Li environment as well as the Li-ion dynamics. NMC811 is a challenging material for such studies due to the high number of paramagnetic centres (Ni$^{2+}$, Ni$^{3+}$, Mn$^{4+}$), leading to short relaxation times and large hyperfine interactions. The acquisition and interpretation of $^7$Li NMR spectra of NMC811 will be demonstrated in this contribution, including data acquired on ex situ and in situ samples.

Ex situ measurements enable the acquisition of NMR spectra under fast magic-angle spinning which yields considerably improved spectral resolution. The ex situ $^7$Li NMR spectra taken on NMC811 cathodes at different states-of-charge (SOC) reveal a strong increase of Li-ion hopping rates during charge which is confirmed by variable temperature measurements.$^{[1]}$ Modelling of these spectra allows estimating the hopping rates and also reveals that Li mobility decreases drastically at high SOC, which is accompanied by Li/vacancy ordering.$^{[1]}$

In situ $^7$Li NMR measurements on NMC811/graphite full-cells are used to simultaneously monitor Li ions in different parts of the cell such as the cathode, the anode, and the electrolyte. We will show a series of measurements at different charging rates and at different temperatures, providing real-time insights into the processes during electrochemical cycling in the whole cell and their contributions to degradation.

[1] K. Märker, P. J. Reeves, C. Xu, K. J. Griffith, C. P. Grey, Chem. Mater. 2019, 31 (7), 2545–2554.

Speaker: Dr Katharina Märker (University of Cambridge; The Faraday Institution)
• 12:30
Surface Structure Determination of Heterogeneous Catalysts by DNP SENS 30m

Dynamic Nuclear Polarization (DNP) is one of the promising approaches to overcome the sensitivity limitations of solid-state NMR, and has recently emerged as a powerful technique to amplify the NMR signals of surface species.1 We have recently demonstrated that the three-dimensional (3D) structure of a model organometallic platinum complex anchored on an amorphous silica can be fully determined by combining DNP Surface enhanced NMR spectroscopy (DNP SENS) with EXAFS data.2 Here we extend this approach to determine the surface structure of a catalyst containing iridium (Ir) N-heterocyclic carbene (NHC) active sites grafted onto a silica surface.3 We will first present the 3D structure of the NHC precursor, obtained from 10 internuclear distances, measured from REDOR experiments. This NHC precursor is partially converted into a Ag-NHC intermediate and then to the final Ir-NHC complex, which results in a mixture of different surface species. We will then demonstrate strategies to overcome this challenge, and get access to targeted structural insights on a surface containing multiple well-defined sites. In particular, the implementation of selective REDOR allowed the measurement of several non-ambiguous, long-range 29Si-{13C} distances. They clearly indicate that the structure of the Ag-NHC and Ir-NHC differs from that of the precursor, but also from the Pt complex determined previously. The NMR and EXAFS data point towards the presence of residual organic ligands coordinating the metal center. Establishing fine relationships between the structure and activity of a catalyst is essential in order to develop systems with improved efficiency.

1. Berruyer, P. et al, eMagRes. 2018, 7, 93–104.
2. Berruyer, P. et al, J. Am. Chem. Soc. 2017, 139, 849–855.
3. Romanenko, I. et al, Angew. Chemie Int. Ed. 2015, 54, 12937–12941.
Speaker: Ribal Jabbour (High-Field NMR Center, Université de Lyon, FRE 2034, CNRS/ENS Lyon/ UCB Lyon1)
• 13:00 14:00
Lunch 1h Harnack House

Harnack House

• 14:00 16:00
Posters: odd numbered presentations Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

• 16:00 16:15
Coffee 15m
• 16:15 17:40
Biomolecules: Phase Separation: Session 18 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 16:15
Conformations of Tau in Dynamic Assemblies 35m

The microtubule‐associated protein Tau plays a key role in Alzheimer’s disease (AD). In healthy conditions, Tau binds to tubulin and microtubules, promotes tubulin polymerization and regulates microtubule dynamics in neurons. However, during the course of AD, Tau aggregates into oligomers and amyloid fibrils, which further associate into neurofibrillary tangles in the intracellular space. The appearance and distribution of Tau aggregates correlates with the loss of neurons and cognitive functions in AD.

We use NMR spectroscopy in combination with other biophysical tools to study the structure and dynamics of Tau in different physiological and pathological states. I will report on our recent findings regarding:
- Liquid-liquid phase separation of Tau
- Molecular recognition of Tau by the human Hsp90 chaperone system

(1) Oroz J, Kim JH, Chang BJ, Zweckstetter M. Nat Struct Mol Biol. 2017 24:407-413.
(2) Oroz J, Chang BJ, Wysoczanski P, Lee CT, Pérez-Lara Á, Chakraborty P, Hofele RV, Baker JD, Blair LJ, Biernat J, Urlaub H, Mandelkow E, Dickey CA, Zweckstetter M. Nat Commun. 2018 9:4532.
(3) Ambadipudi S, Biernat J, Riedel D, Mandelkow E, Zweckstetter M. Nat Commun. 2017 8:275
(4) Ambadipudi S, Reddy JG, Biernat J, Mandelkow E, Zweckstetter M. Chem Sci. 2019 DOI: 10.1039/c9sc00531e
(5) Ukmar-Godec T, Hutten S, Grieshop MP, Rezaei-Ghaleh N, Cima-Omori M-S, Biernat J, Mandelkow E, Söding J, Dormann D, Zweckstetter M. Nat Commun. 2019 in press

Speaker: Prof. Markus Zweckstetter (German Center for Neurodegenerative Diseases (DZNE))
• 16:50
Multivalency and phase separation of measles virus replication machinery. 25m

RNA viruses tend to concentrate their replication machinery within so called “viral factories”. This membraneless compartment, formed by phase separation, has liquid properties and has been shown for several viruses in vivo [1,2]. Measles virus (MeV) is the cause of measles, it infects T-cells and macrophage cells, belongs to Paramyxoviridae family. Its genome consists of non-segmented negative-strand RNA, which encodes eight proteins from six genes (N, P, L, H, F and M). The MeV replication machinery consists of viral RNA covered by nucleoproteins (N), and protecting the genome against the host cell immune system, Large protein (L), the viral RNA-polymerase, and its essential co-factor, Phosphoprotein (P) [3]. As has been shown for other single strand RNA viruses there are N and P are essential for phase separation [1,2]. As shown by NMR, N and P interact with each other via long intrinsically disordered domains during their viral cycle [4,5].
Here we show for the first time phase separation of MeV N and P proteins in vitro, identify phase separation scaffold and characterise its liquid behaviour. Using NMR we describe multiple interactions between P and N disordered regions which are essential for phase transition and regulation of its biophysical parameters. In addition, we could show a non-stationary stoichiometry between N and P within droplets and propose the model on the basis of observations.
[1] Brunel J. et al., J Virol., 2014, 88(18):10851-63
[2] Nikolic J. et al., Nat Com, 2017, 8(58):00102-9
[3] Heinrich B. et al., mBio, 2018, 9(5):02290-17
[4] Jensen et al., PNAS, 2011, 108(24):9839-44
[5] Milles et al., Sci Adv, 2018, 22;4(8):eaat7778

Speaker: Serafima Guseva (Viral Replication Machines Group & Protein Dynamics and Flexibility by NMR Group, Institut de Biologie Structurale (IBS), CEA, CNRS, University Grenoble Alpes, Grenoble, France)
• 17:15
A Molecular View of the Liquid to Gel Phase Transition of Heterochromatin Protein HP1α 25m

Heterochromatin protein 1α (HP1α) plays a central role in the organization of nuclear content and in the regulation of gene expression and chromatin compaction. Recent work has shown that this protein can phase separate into liquid droplets and gels in vitro, properties that have vast implications for the mechanism of heterochromatin formation and regulation in cells. Despite the tremendous amount of interest in this process, the amorphous, dynamic and viscous nature of the gel condensates has precluded high-resolution analysis of the molecular interactions that underlie HP1α transitions. Using magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, here we present for the first time a molecular description of the liquid to gel transition of phosphorylated HP1α. This methodology has allowed us to follow in real time the rigidification of the molecular interaction network during gelation and to identify specific residues that contribute to gel formation. Furthermore, the addition of physiologically relevant chromatin polymers disrupts the gelation process while preserving the conformational dynamics within individual HP1α molecules. Our results suggest an important role for chromatin in determining the material properties of HP1α condensates and in establishing the complex dynamics within heterochromatin compartments. Our methodology can be applied to other protein systems that undergo phase separation and thus provide atomic resolution details of an elusive biological process.

Speaker: Prof. Galia Debelouchina (University of California, San Diego)
• 16:15 17:40
EPR: Session 17 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 16:15
Studying structure and function of sialic acid TRAP transporters from pathogenic bacteria by pulsed EPR, FRET and X-ray crystallography 35m

Many pathogens such as Vibrio cholerae use tripartite ATP-independent periplasmic (TRAP) transporters to scavenge N-acetyl- neuraminic acid (sialic acid) from host organisms. The sialic acid is then incorporated into the bacterial cell wall, as a disguise to protect against detection by the human immune system. TRAP transporters are a structural and functional mix between ABC transporters and secondary active transporters. The substrate binding proteins (SBP) of TRAP transporters are the best studied component and are responsible for initial high-affinity substrate binding. To better understand the dynamics of the ligand binding process, pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy and FRET were applied to study the conformational changes in the N-acetylneuraminic acid-specific SBP VcSiaP. The protein is the SBP of VcSiaPQM, a sialic acid TRAP transporter from Vibrio cholerae. Spin-labeled double-cysteine mutants of VcSiaP were analyzed in the substrate-bound and -free state and the measured distances were compared to crystal structures of the labelled protein. The data were compatible with two clear states only, which are consistent with the open and closed forms seen in TRAP SBP crystal structures. Substrate titration experiments demonstrated the transition of the population from one state to the other with no other observed forms. Mutants of key residues involved in ligand binding and/or proposed to be involved in domain closure were produced and the corresponding PELDOR experiments reveal important insights into the open-closed transition. Further, PELDOR distance measurements on the whole transporter in lipid nano discs were used to evaluate molecular models of the transporter.

Speaker: Dr Gregor Hagelueken (University of Bonn)
• 16:50
When stronger magnets don’t help: Methods for disentangling overlapping high-field EPR spectra illustrated in record organic solar cell blend PBDB-T:ITIC 25m

Spectral overlap, even at high field, is a problem generally encountered in many EPR studies. In the specific case of bulk-heterojunction (BHJ) organic solar cells (OSCs), the paramagnetic species of interest are light-induced radicals which are created as a pair after charge transfer at the interface between the donor polymer and molecular acceptor regions making up the BHJ blend. Hence, the similar g-values expected for the positive and negative organic radicals often lead to strong spectral overlap complicating the unambiguous assignment of the light-induced (LI) EPR spectrum.

The donor-acceptor combination studied here, PBDB-T:ITIC, was the first fullerene-free OSC to recently achieve >11% efficiency, challenging the state-of-the-art polymer-PC$_{71}$BM devices [1]. For this blend, the two-component structure of the LI-EPR spectrum could not even be resolved at W-band frequency (94 GHz). Therefore we separated the two contributions to the total EPR spectrum by exploiting two different properties of the charge-transfer radicals, namely the (small) difference in their longitudinal ($\text{T}_1$) relaxation times and the presence of a unique magnetic nucleus, $^{14}$N, in ITIC. For the $\text{T}_1$-based method, we applied an inversion-recovery filter to selectively suppress one component in the spin echo analogously to the relaxation-filtered hyperfine spectroscopy (REFINE) technique first proposed by Maly et al. [2]. Sensitive detection of the $^{14}$N hyperfine couplings at W-band frequency was achieved by means of electron-electron double resonance (ELDOR)-detected NMR (EDNMR). Here we demonstrate the application of EDNMR-induced EPR to obtain an EPR spectrum containing only contributions from the ITIC radical [3]. Both approaches are validated by LI-EPR spectra on related blends which yield better-resolved spectra of the individual PBDB-T and ITIC radicals.

[1] W. Zhao et al., Adv. Mater., 2016, 28, 4734.
[2] T. Maly, T. F. Prisner, J. Magn. Reson., 2004, 170, 88.
[3] M. Van Landeghem et al., J. Magn. Reson., 2018, 288, 1.

Speaker: Ms Melissa Van Landeghem (Department of Physics, University of Antwerp)
• 17:15
Non-magnetic magnetic resonance 25m

Magnetic resonance observes spin transitions whose frequencies depend on magnetic field because spin is associated with magnetic moment. Allowed transitions involve a unit change of the magnetic quantum number. If the magnetic quantum number is not a good quantum number, other transitions can be partially allowed. The transition moment of such “forbidden” transitions depends on magnetic field. All this is taken for granted. Yet, in three-pulse electron spin echo envelope modulation (ESEEM) experiments on a Mn(II)-doped [(CH3)2NH2][Zn(HCOO)3] metal-formate framework, we have recently observed features whose frequencies and normalized intensities did not change in the magnetic field range between 0.325 and 3.35 T [1]. By a series of isotope substitution experiments these signals could be attributed to the methyl groups of the dimethylammonium cation. Here we demonstrate that they derive from hyperfine-perturbed methyl tunnel splitting. We discuss how a nearby electron spin breaks C3 symmetry of the methyl group and why this can lead to polarization transfer to the tunnel splitting transition. We show that, by manipulating solely the electron spin by microwave pulses, the tunnel-split states can be coherently superimposed and the phase of this coherence can again be read out by the electron spin. Spectra and signal buildup are in good agreement with simulations, whereas rotation barriers derived from the tunnel splitting for two frameworks are in fair agreement with predictions by density functional theory. We discuss the possibility of observing the hyperfine-unperturbed tunnel splitting in an ESEEM experiment and the potential of the effect for dynamic nuclear polarization.
Reference: [1] Simenas, M., Macalik, L., Aidas, K., Kalendra, V., Klose, D., Jeschke, G., Maczka, M., Volkel, G., Banys, J. & Poppl, A. Pulse EPR and ENDOR Study of Manganese Doped [(CH3)2NH2][Zn(HCOO)3] Hybrid Perovskite Framework. J. Phys. Chem. C 121, 27225-27232 (2017)

Speaker: Prof. Gunnar Jeschke (ETH Zürich)
• 16:15 17:40
Instrumentation: Session 19 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 16:15
Dissolvable inserts for achieving performance enhanced resonators 35m

Dissolvable 3D-printed templates can be used to produce NMR transceiver coils of a particular geometry. Wire coils such as variable-pitch solenoids are wound on dissolvable 3D printed forms, which are then dissolved away in solvent. The use of high-temperature resin, with the appropriate solvent, enables annealing to be performed before dissolving the template. This approach allows facile, reproducible production of coils even in the hands of inexperienced researchers. The ability to design a coil and its corresponding template in a CAD program, simulate the magnetic and electric fields in its vicinity, print out the template, and make it greatly facilitates the process of testing coil designs and sharing successful ones with other laboratories. This approach can also be used to enable production of more complicated designs that are not easily made with hand winding. I will describe strategies for using 3D printed coil forms in conjunction with simulation-based optimization to produce high-homogeneity rf coils. Simulations and experimental (benchtop and NMR) measurements using different resonator designs will be discussed, along with future applications to biomolecular NMR.

Speaker: Prof. Rachel Martin (University of California, Irvine)
• 16:50
Rapid scan EPR-on-a-chip 25m

Electron paramagnetic resonance (EPR) is the method of choice to investigate and quantify paramagnetic states in e.g. semiconductor devices, proteins, catalysts and molecular nanomagnets. Common EPR spectrometers use microwave (mw) resonators, where the sample is inserted. This design, however, limits the versatility for in situ / operando measurements. Here, we present an improved design of a miniaturised EPR spectrometer, implemented on a single microchip (EPR-on-a-chip). Instead of an mw resonator, an array of coils, each from a voltage-controlled oscillator (VCO), with a diameter of a few hundred micrometer is used simultaneously as mw source and detector, replacing the entire microwave bridge. Similar to EPR microresonators, the filling factor of the EPRoC is high by design, leading to a better absolute spin sensitivity than conventional EPR. The usage of the VCO allows to sweep the microwave frequency, instead of magnetic field as in the conventional EPR, thus enabling operation with a permanent magnet. Due to its compactness, EPRoC can be incorporated into conventional thin-film growth reactors, (electro)chemical cells, batteries or in UHV environments.

Frequency sweeps in combination with the intrinsically high B1 render EPRoC perfect for rapid frequency scan EPR (rsEPRoC) with scan rates up to 2 000 THz/s. Rapid scan EPR can lead to a signal-to-noise improvement especially for samples with long relaxation times, which would otherwise be saturated in continuous wave EPR. We demonstrate the increased sensitivity of rsEPRoC, by investigating a few micrometer thick layers of amorphous silicon (a-Si) on quartz.

In this talk, we will review the recent advances in rsEPRoC, show first results on amorphous silicon samples, and discuss applications that will benefit from the increased sensitivity.

Speaker: Silvio Künstner (Berlin Joint EPR Laboratory, Institut für Nanospektroskopie, Helmholtz-Zentrum Berlin für Materialien und Energie)
• 17:15
Rheology and 23Na Multiple Quantum Filtered (MQF) rheo-NMR and MRI of Bile Salt Micelles 25m

Bile or gall is a dark green to yellowish brown fluid, produced by the liver of most vertebrates and aids the digestion of lipids in the small intestine. The composition of bile is mostly water (97%) however, it also contains small amount (0.7%) of bile salts, as well as fats and inorganic salt ions. Bile salts are complex molecules that tend to form micellar aggregates in solutions if their and/or salt concentration increases. This process affects rheology of the bile thus making its viscosity shear-dependent during flow in a bile duct. Pathological bile extracted from gallbladder and liver patients is non-Newtonian [1], and is hypothesized that the formation of micellar aggregates in the bile during flow through the duct contributes to this behaviour. This currently is being explored for potential clinical benefits for the use in 23Na whole body MRI. One of the principal components of human bile is taurodeoxycholic (TDC) acid that forms a sodium salt, NaTDC, in the excess of Na+ [2]. We studied temperature and shear effects on the micellar formation in 0.2M NaTDC /0.25M NaCl and in 0.2M NaTDC /0.5M NaCl systems using rheology and 23Na MQF rheo-NMR and MRI. Double quantum filtered magic angle (DQF MA) and triple quantum filtered (TQF) rheo-NMR [3-5] was performed at multiple shear rates and temperatures. The formation of shear-induced phase was clearly demonstrated by 23Na rheo-NMR and it was found that 23Na MQF rheo-NMR methods were able to detect and characterise the formation of the shear–induced phase more efficiently than bulk rheometry methods. 23Na MRI with MQF filters allowed to map zones where shear-induced phase was formed and to characterise molecular alignment in the gap. This demonstrates the potential of 23Na MQF MRI contrast for the in-vivo molecular mechanics for clinical benefits.

Speaker: Dr Galina Pavlovskaya (University of Nottingham)
• 16:15 17:40
NMR in Drug Design: Session 20 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 16:15
Cancer Drug Discovery Using Fragment-Based Methods and Structure-Based Design 35m

Cancer is a devastating disease that affects the lives of almost everyone, and its effective treatment still remains an important unmet medical need. In order to discover new cancer drugs, we are applying fragment-based methods and structure-based design to identify and optimize small molecules that inhibit highly validated cancer targets. Although many of these targets are technically challenging and thought to be undruggable, fragment-based methods offer several advantages over more conventional approaches which suggest that it may be possible to achieve success. In this presentation, examples will be given of how this methodology can be used to discover small molecules that bind to highly validated but technically challenging cancer targets.

Speaker: Prof. Stephen Fesik (Vanderbilt University School of Medicine)
• 16:50
Modulation of aggregating proteins studied by NMR and beyond in neuro- and cellular degeneration 25m

In Parkinson’s disease (PD), a-Synuclein aggregates to Lewy bodies, which are connected to neuronal dysfunction and death, similar to Abeta and tau in Alzheimer’s (AD), prion protein in Creutzfeldt Jacob and IAPP in Type II diabetes mellitus (T2DM). Using structural biology derived predictions (1), a-Synuclein is shown to form non-toxic intrinsically disordered monomers and non-toxic fibrils, while immediate toxicity is exerted by oligomers (2). Prevention of formation of these toxic oligomers by small molecules, specifically anle138b, observed in vitro and in vivo (3) using ultracentrifugation or superresolution imaging, specifically anle138b, leads to neuroprotection and restoration of functionality of the neurons in all mentioned diseases, specifically in PD (3), MSA (4), AD based on tau (5) or Abeta42 overexpression (6) and T2DM (7). Biophysical characterization of anle138b with target proteins will be discussed (5,8) in solution and in membranes with NMR spectroscopy.

1) Bertoncini, CW; et al. PNAS, 102 (5): 1430-1435
2) Karpinar, DP; et al. Embo Journal, 28 (20): 3256-3268
3) Wagner, J; et al. Acta Neuropathol. 125, 795-813 (2013); Levin, J; et al. Act. Neuropath. 127, 779-780 (2014); Giese, A; Bertsch, U; Kretzschmar, H; Habeck, M; Hirschberger, T; Tavan, P; Griesinger, C; Leonov, A;, Ryazanov, S; Weber, P; Geissen, M; Groschup, MH; Wagner, J. (2010) WO/2010/000372; A. Giese, F. Schmidt, C. Griesinger, A. Leonov, S. Ryzanov: WO2017/102893; Wegrzynowicz M et al. Acta Neuropathol.https://doi.org/10.1007/s00401-019-02023-x (2019)
4) Heras-Garvin, A. et al. Movement Disorders in press
5) Wagner, J. et al. Act. Neuropath.130, 619-631 (2015);
6) Martinez Hernandez, A; et al. EMBO Mol. Med. 10, 32-47 (2018)
7) J. Hoppener et al. “Glucose and HbA1c normalization and insulin sensibilization in a HIAPP overexpression model in T2DM” in preparation
8) Deeg, A.A. et al. Biochim. Biophys. Act. 1850 (9), 1884-1890 (2015); Reiner, A.M. et al. Biochim. Biophys. Acta Gen. Subj. 1862, 800-807 (2018)

Speaker: Prof. Christian Griesinger (MPIbpc)
• 17:15
NMR as a tool for defining cyclotide membrane binding: applications in medicine and agriculture 25m

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia

Naturally occurring as well as ‘designer’ cyclic peptides offer great potential as leads for drug design or crop protection agents in agriculture. This talk will focus on one class of cyclic peptides known as cyclotides, which are topologically unique in that they have a head-to-tail cyclized peptide backbone and a cystine knotted arrangement of three conserved disulfide bonds. This makes cyclotides exceptionally resistant to chemical, thermal or enzymatic degradation and, indeed, cyclotides are amongst nature’s most stable proteins. They occur in plants from the Rubiaceae (coffee), Violaceae (violet), Solanaceae (nightshade), Fabaceae (legume) and Cucurbitaceae (cucumber) families of plants where their natural function is presumed to be in host defence. This presentation will describe the membrane binding properties of cyclotides and how the delineation of these properties by NMR and other biophysical techniques has assisted in the understanding of their natural defense functions and pharmaceutical applications. In particular, solid phase synthesis has allowed us to make a range of modified cyclotides to probe structure-activity relationships. A cyclotide-containing product was recently approved for insect control in cotton and macadamia nut crops, marking the first commercial application of cyclotides in agriculture, and there are more than two dozen published examples of cyclotide-based drug leads.

Acknowledgments: Work in our laboratory is supported by the Australian Research Council and the National Health & Medical Research Council

Speaker: Prof. David Craik (University of Queensland)
• 16:15 17:40
Small Molecules: Session 16 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 16:15
New approaches for J-coupling measurement and five-membered ring conformation analysis 35m

For any conformational analysis based on spectroscopic data, both the amount of data extracted and the accuracy of the theoretical relation between data and conformation are key. In this presentation, new developments for both the measurement of 1H-1H J-couplings and their use for conformational analysis of five-membered rings will be discussed.
Measuring J-couplings in compounds that are mostly aliphatic can be problematic, as the limited chemical shift dispersion and the presence of many couplings often results in intricate and overlapping multiplets. Also strong coupling conditions are common, which complicates accurate J-coupling extractions. A number of strategies will be presented that build on the PSYCHEDELIC experiment [1] that can resolve these issues.
Five-membered rings are widely occurring in biological and synthetic compounds, and the preferred ring conformation is often crucial for function. In contrast to six-membered rings, five-membered rings are generally flexible, sampling a distribution of conformers, making their analysis challenging. Experimentally, vicinal J-couplings are ideal for conformational analysis of ring systems, as they are sensitive to dihedral angles. However, a key issue is that the available Karplus relations are not always appropriate for a given system. For instance, for difluorinated prolines [2,3], existing Karplus relations for 1H-1H couplings turn out to be insufficently accurate, while no appropriate relations exist for 1H-19F couplings. Also non-bonded electronic interactions across the five-membered ring have significant impact on the J-coupling. We therefore resorted to an alternative approach that is based on extensive mapping of the full conformational space of model compounds using DFT, followed by J-coupling calculation.
[1] Sinnaeve D. et al., Angew. Angew. Chem. Int. Edit. (2016), 55, 1090
[2] Hofman G.J. et al., Chem. Commun. (2018), 54, 5118
[3] Hofman G.J. et al., J. Org. Chem. (2019), 84, 3100

Speaker: Dr Davy Sinnaeve (CNRS / Université de Lille)
• 16:50
A boost in Drug Discovery with Secondary-labeled Hyperpolarized Ligands 25m

Hyperpolarization by dissolution DNP[1] provides a route to enhancing 13C MR sensitivity by more than four orders of magnitude on a wide range of small molecules. However, many potential applications of dDNP (metabolomics, drug discovery, etc.) would highly benefit from a higher efficiency, throughput, and repeatability of the method.

In this context, we had demonstrated in 2016 that high levels of polarization were reachable in short times (60% in 8 min)[2] and with high repeatability (CV=3.6%)[3].

One important drawback still remains in dDNP which is that it generally relies on low natural abundance 13C spins (1.1%). In 2009, Wilson et al. proposed an approach where amine groups in amino acids were labeled with [1,1-13C] acetic anhydride[4], and subsequently hyperpolarized. Though very promising, this approach had not been taken up by the dDNP community till 2018 where we revisited this secondary labeling approach in the context of NMR drug screening[5]. We have showed how ligands could be secondary labeled, used to probe interactions with target proteins through 13C NMR.

We are now working at combining this concept with dDNP with the aim of decreasing experiment time and sample concentration by orders of magnitudes compared to the classical approach (STD / WaterLOGSY). This has the potential to considerably improve the detection and identification of ligands, and shorten the discovery time of new drug candidates.

References

[1]. Ardenkjaer-Larsen, J. H. et al. Proc. Natl. Acad. Sci. USA 100, 10158–10163 (2003)
[2]. Bornet, A. et al. Phys. Chem. Chem. Phys. 18, 30530–30535 (2016)
[3]. Bornet, A. et al. Anal. Chem. 88, (2016).
[4]. Wilson, D. M. et al. Proc. Natl. Acad. Sci. 106, 5503–5507 (2009)
[5]. Cala, O. et al. Euromar 2018 Nantes France

Speaker: Dr Olivier Cala (Université de Lyon, Université Claude Bernard Lyon 1, ENS de Lyon, CNRS, CRMN FRE 2034)
• 17:15
Monitoring Oxygen Levels in Microfluidic Devices using 19F NMR 25m

We report an in-situ, non-invasive approach to quantify oxygen partial pressure
in microfluidic lab-on-a-chip (LoC) devices. LoC systems provide a versatile
platform to culture biological systems. As they allow a detailed control over
the growth conditions, LoC devices are finding increasing applications in the
culture of cells, tissues and other biological systems [1]. Integrated
microfluidic NMR spectroscopy [2] allows non-invasive monitoring of metabolic
processes in such systems. Quantification of oxygen partial pressure would help
ensuring stable growth conditions, and provide a convenient means to
assess the viability of the cultured system. However, oxygen, one of the most
important metabolites, cannot be quantified using either proton or carbon NMR
spectroscopy.
As is well known, the oxygen partial pressure can be determined by MRI in vivo
by measuring the 19F spin-lattice relaxation time of perfluorinated agents [3].
Here, we show that the oxygen partial pressure in microfludic devices of 2.5 µl
can be quantified using the 19F spin-lattice relaxation rate of perfluorinated
tributylamine. The compound is added to the aquous perfusion
medium in the form of micrometer-sized droplets. Our set up comprises a
microfluidic device and a PDMS layer sandwiched between two 3D printed holders.
The droplet emulsion is delivered via a syringe pump and carbogen is delivered
through a separate channel. The semi- permeable PDMS layer acts as a diffusion
bridge between the liquid and gas channels, allowing for oxygen to diffuse into
the emulsion. T1 is obtained through standard inversion recovery experiments
detected using a home-built transmission-line probe.[2] Due to the non-toxic nature of droplet emulsion, it can be easily incorporated into the perfusion fluid
allowing for quantification of tissue oxygen levels.
1. Gracz et al., Nature Cell Biology 17, 340–349, 2015
2. M.Sharma, M.Utz, J.Mag.Res 303, 75-81, 2019.
3. R.Manson et al., Magnetic Resonance in Medicine 18, 71-79, 1991.

Speaker: Ms Sylwia Ostrowska (University of Southampton)
• 17:40 18:25
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 17:45
Transporter Conformational Dynamics from Spin Labeling EPR Spectroscopy 40m

Understanding the mechanisms of membrane proteins entails complementing static structures with the conformational changes in the structure. Recent advances in Double Electron-Electron Resonance (DEER) spectroscopy along with computational methods to generate restrained models of proteins are enabling unprecedented insights into the conformational dynamics of active transporters. My laboratory use EPR methods to define conformational equilibria that mediate the transport cycle of ion-coupled symporters and antiporters as well as ABC transporters. I will describe comparative analysis of ATP-binding cassette (ABC) exporters that reveals the role of dynamics in their transport cycles that resolve long standing question in the field . Together, these studies are illuminating the mechansitic commonalities and differences in the family.

Speaker: Prof. Hassane S. Mchaourab (Vanderbilt University)
• 18:25 18:55
Society Meetings: AMPERE General Assembly Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 19:00 21:00
Posters: even numbered presentations Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

• Wednesday, 28 August
• 08:40 10:00
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 08:40
Recent Developments of DNP Enhanced Solid-State NMR Spectroscopy at High Magnetic Field and Fast MAS 40m

Dynamic Nuclear Polarization (DNP) has recently evolved into a cornerstone technology to overcome the sensitivity limitations of solid-state NMR. This technique, originally developed for low magnetic fields, has been shown to be applicable at high fields, opening new avenues in materials and life sciences. In this presentation we will review some recent advances from high magnetic field (18.8 T) and fast Magic Angle Spinning (MAS) (~ 40 kHz) DNP NMR. In particular, we will present our efforts towards the development of polarizing agents tailored for efficient DNP at high fields and fast MAS. Applications to the characterization of challenging catalytic surfaces and of biological assemblies will also be presented.

Speaker: Prof. Anne Lesage (University of Lyon)
• 09:20
Boosting clinical diffusion MRI with principles from solid-state and Laplace NMR 40m

Diffusion MRI is today used in clinical routine for detecting stroke and grading prostate tumors, as well as in clinical research studies of for instance neurological diseases and normal brain development. The overwhelming majority of the diffusion MRI measurements are performed with motion encoding by the most basic form of the pulsed-gradient spin echo sequence from the mid-60s, which is sensitive to local diffusivities, restrictions, anisotropy, flow, and exchange. While it may be convenient to have a single experiment to detect a wide range of different diffusion properties, the lack of selectivity becomes a nuisance when attempting to assign the experimental observations for a complex, heterogeneous, and anisotropic material like the living brain to a specific diffusion mechanism. This lecture will give an overview of our recent work in redesigning diffusion MRI using principles that are well known in multidimensional solid-state NMR spectroscopy and low-field NMR of porous materials. The key features of this new “multidimensional diffusion MRI” approach are gradient waveforms targeting specific motion mechanisms and multidimensional acquisition and analysis protocols wherein the different mechanisms are separated and correlated. The gradient waveforms are often borrowed from classical sample reorientation techniques such as magic-angle spinning, variable-angle spinning, and double rotation. Data inversion is performed with algorithms from multidimensional Laplace NMR, in particular the more sophisticated Monte Carlo inversion generating ensembles of plausible distributions and estimates of the uncertainties of the obtained distributions and scalar parameters. Clinical application examples include studies of microstructure in meningioma and glioma brain tumors as well as white matter degeneration in multiple sclerosis.

Speaker: Prof. Daniel Topgaard (Lund University)
• 10:00 10:30
Coffee 30m
• 10:30 12:30
Biomolecules: Integrated Structural Biology: Session 23 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 10:30
Integrative structural biology of non-coding RNA-protein complexes: telomerase and 7SK 35m

Telomerase and 7SK are essential eukaryotic RNA-protein complexes (RNPs) involved in telomeric DNA synthesis and regulation of mRNA transcription, respectively. Each comprises a non-coding RNA and constitutively and/or transiently associated proteins. Telomerase maintains the DNA at the ends of linear chromosomes, thereby preventing genomic instability. Its catalytic core is a non-coding telomerase RNA (TER) and a unique telomerase reverse transcriptase (TERT); other associated proteins are involved in biogenesis, assembly, recruitment to telomeres, and recruitment of other proteins of the DNA synthesis machinery. TERT uses a template complementary to ~1.5 telomere repeats in TER to repetitively synthesize the telomere repeat sequence at the 3’ end of the DNA (TTGGGG in ciliates, TTAGGG in vertebrates), but this template alone is insufficient for activity with TERT. Multiple steps of single-stranded telomeric DNA template binding/realignment, nucleotide addition, strand separation, and template translocation are required for synthesis of a single telomere repeat and telomere repeat addition processivity (RAP). We have been using an integrative structural biology approach combining NMR spectroscopy, X-ray crystallography, mass spectrometry, and electron microscopy to study the structure and function of telomerase from the ciliate Tetrahymena and from humans. I will discuss how our NMR studies of telomerase RNA structure and dynamics have been combined with cryo electron microscopy to help elucidate the roles of TER and proteins in this remarkable enzyme. I will also present recent NMR and X-ray crystallography results on the human long-noncoding RNA 7SK and two of its protein partners Larp7 and methylphosphate capping enzyme (MePCE).

Speaker: Prof. Juli Feigon (University of California Los Angeles)
• 11:05
A Case of Domain Cooperation in a Multidomain Protein Interaction at Telomeres. 25m

Telomeres are specialized structures located at the ends of linear chromosomes essential for cell viability and genome integrity. Their protective function is due to the formation of the Shelterin complex, that caps the end of the DNA. In the mammalian complex, Telomeric Repeat-binding Factor 2 (TRF2) interacts with TRF2-interacting protein 1 (RAP1).
We previously showed by ITC that TRF2-RAP1 interaction involves a complex biphasic mechanism [1]. RAP1, as TRF2, is a multidomain protein, comprising a N-terminal domain with sequence similarity to BRCT domains (RAP1[BRCT]), a central pseudo-Myb domain and a C-terminal domain that binds with high affinity with the so-called RAP1-binding domain of TRF2. The function of the RAP1[BRCT] is not yet assigned.
Here we report a structural analysis of the binding between the different domains of RAP1 and TRF2. First, we solved the solution structure of RAP1[1-114] that belongs to BRCT domain family. The conformation of the YRLGP sequence in RAP1[1-114] is well defined and similar to that adopted in a 14-mer peptide crystallized in complex with the dimeric TRFH domain of TRF2 [1]. This led us to build a RAP1[1-114]-TRFH dimer model. However, we showed by NMR that the isolated RAP1[1-114] domain is not able to interact with TRFH. This observation seemed a priori in contradiction with ITC data previously obtained to characterize the interaction of the full form of RAP1 with TRF2. However, re-examination of ITC and NMR interaction data obtained with truncated forms of the two proteins permitted to reconcile these data in the light of the divalent thermodynamics interaction model [3]. The cooperative mechanism revealed by this analysis could contribute to regulate the interaction of TRF2 with partners interacting via YRLGP-like peptides.

[1] Gaullier G., S. Miron, S. et al. (2016). Nucleic Acids Research 44: 1962.
[2] Kane, R., S. (2010). Langmuir 26:8636.

Speaker: Dr Philippe Cuniasse (Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Saclay)
• 11:30
Solution structure of Upstream-of-N-Ras, a 116 kDa multi-domain RNA binding protein 25m

The protein Upstream-of-N-Ras (Unr) is a highly conserved and abundant RNA binding protein with elevated expression levels in several cancer types. Here, it is supposed to bind certain mRNAs to regulate their translation. In Drosophila, Unr acts an RNA chaperone, where it binds to long non-coding RNA RoX2 to promote formation of the dosage compensation complex. In all hitherto publications concerning Unr it has been proposed that it features five cold shock domains (CSDs), which are known for binding single-stranded RNAs. These domains are presumably connected with flexible linkers.
However, our construct optimizations and NMR analysis revealed the presence of altogether nine CSDs. Interestingly, the predicted canonical CSDs are interspersed with novel non-canonical CSDs, which share the same fold with the canonical ones, but lack their otherwise conserved trademark RNA binding residues. Structure determination of several tandem and triple domain constructs by NMR and X-ray crystallography revealed that there are no flexible linkers and that the domains have indeed a fixed orientation towards each other. This indicates that Unr, although binding to single-stranded RNAs via its CSDs, achieves also RNA structure specificity due to the fixed orientations and distances of canonical CSDs.
Complementing the structures with small-angle X-ray scattering and further NMR data we were able to obtain a high-resolution structural ensemble of the full-length protein. Additional mutational analysis using biochemical, biophysical and cellular assays confirm the importance of inter-domain arrangements for RNA specificity and function.
This study presents a new paradigm of how a general single-stranded RNA binding protein achieves RNA structure specificity.

Speaker: Dr Janosch Hennig (Structural and Computational Biology Unit, EMBL Heidelberg)
• 11:55
High molecular-weight complexes in the regulation of gene expression: a view by integrative structural biology 35m

The Regulator of Ty1 Transposition protein 106 (Rtt109) is a fungal histone acetyltransferase required for histone H3 K9, K27 and K56 acetylation. These acetylation sites have been linked to processing and folding of nascent H3 and play an integral role in replication- and repair-coupled nucleosome assembly. Rtt109 is unique in its activation, performed by two structurally unrelated histone chaperones, Asf1 and Vps75. These proteins stimulate Rtt109 activity via different mechanisms. Rtt109 - Asf1 association has been proposed to be responsible for K56 acetylation, while the Rtt109-Vps75 interaction is required for K9 acetylation.

In our work we find that Rtt109, Vps75 and Asf1 are capable of assembling as a previously uncharacterized complex onto the substrate H3-H4 dimer. Using an integrative structural biology approach based on a powerful combination of solution state NMR and small angle neutron scattering (SANS) we solve the structure of this complex and provide a structural basis for the efficiency and selectivity of acetylation at the at the H3 K9, K27 and K56 sites.

Speaker: Prof. Teresa Carlomagno (Leibniz Universität Hannover)
• 10:30 12:30
Biomolecules: Molecular Chaperones: Session 24 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 10:30
The True Tales of the Flexible Tails – Interaction of J-domain Protein with Hsp70 chaperones 35m

Hsp70s are ubiquitous chaperones tasked with safeguarding proteins throughout their entire lifecycle, from synthesis to degradation, and are thus critical for maintaining protein-homeostasis. The ATPase cycle of Hsp70s, which is allosterically coupled to the binding and release of their substrates, is, in turn, regulated by a large set of dedicated co-chaperones consisting of nucleotide-exchange-factors and of J-domain proteins. Humans contain multiple such J-domain-proteins(JDPs), all of which interact through their conserved J-domain with Hsp70s in an ATP dependent manner. The most abundant JDPs in the cell belong to very structurally-similar Class-A and Class-B families and contain, in addition to the J‐domain, an adjacent glycine‐rich-region, two client binding domains (CTDI and CTDII), and a dimerization domain.
Despite the many similarities, however, Class-A and Class-B J‐proteins still exhibit significant differences in both structure and function. We were, therefore interested to see whether the two classes of JDPs also display differences in their interactions with Hsp70 chaperones.
To this end, we used solution NMR spectroscopy to test the interaction of Hsc70 with the two classes of DnaJ chaperones, and monitored DnaJ-dependent Hsc70 activation via functional assays.

Our results uncover that Class-A and Class-B DnaJs, in fact, interact with Hsc70 in a different manner. While both classes of DnaJs bind Hsc70 through their J-domain, with this weak interaction activating Hsc70 catalytic activity, Class-B DnaJs also contain an additional Hsc70 binding site. When in complex with Hsc70, we have identified that DnaJB1 interacts both via its N-terminal J-domain and its CTDI substrate-binding-domain. The binding of the Hsc70 C-terminal region to DnaJB1 CTDI causes a conformational change that exposes the DnaJB1 J-domain for Hsc70 binding, with only this second interaction leading to activation of Hsc70 catalytic activity. This, then points to a new regulatory interaction between class-B DnaJ chaperones and Hsc70, that is absent in class-A DnaJs.

Speaker: Dr Rina Rosenzweig (Weizmann Institute of Science)
• 11:05
Dynamic regulation of human Hsp70 chaperone functional cycle by its co-chaperones and client protein 25m

BiP is the only member of the Hsp70 chaperone family in the human endoplasmic reticulum (1). Its chaperone activity is driven by ATP binding and hydrolysis that trigger the conformational change regulating the docking and undocking of its nucleotide-binding domain (NBD) and substrate-binding domain (SBD). To achieve its many functions, BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) and J-domain protein (JDP). Although structure of intermediate states of BiP have been determined by x-ray crystallography (2) and specific segments of its functional cycle have been explored by NMR (3, 4) and FRET studies (5), characterization of the entire mechanism driving BiP function requires studies at the atomic level under working conditions in presence of its co-chaperone network.
Here, we report a study by real-time methyl NMR, under physiological conditions, of the BiP functional cycle in presence of its major cochaperones (JDP and NEF) and a native client protein. We demonstrate that the co-chaperones speed up the BiP functional cycle by tuning the equilibrium between its docked and undocked conformation, via regulating the allosteric communication between NBD and SBD. Furthermore, we reveal how the dynamic network of co-chaperones and client protein regulate BiP activity, providing for the first time at atomic resolution a time-resolved description of the BiP functional cycle. This study opens up new perspectives to understand how dysfunction in the regulation of BiP by its co-chaperones is linked to a broad range of BiP-related diseases such as cancer, cardiovascular and neurodegenerative disease.

References
[1] Wang, J., et al. (2017). Gene 618, 14–23.
[2] Yang, J., et al. (2015). Structure 23, 2191–2203.
[3] Wieteska, L., et al. (2017). eLife 6, 18966.
[4] Rosenzweig, R., et al. (2017). eLife 6, 199.
[5] Rosam, M., et al. (2018). NSMB 25, 90–100.

Speaker: Dr Guillaume Mas (Biozentrum, University of Basel )
• 11:30
NMR Informed Molecular Modeling to Capture Transient Chaperone-Substrate Interactions 25m

Nuclear Magnetic Resonance (NMR) Spectroscopy is a unique tool to study complex systems such as biological macromolecules due to its ability to probe molecular structure and dynamics at atomic resolution and on a wide range of timescales. During the last decades major progresses have been made towards the description of biomolecular dynamics and protein folding. However, fundamental questions remain open.

Chaperone proteins are key elements in regulating protein folding and aggregation prevention. Despite their critical biological role, the mechanisms by which chaperone proteins interact with their substrates remain quite elusive, in part due to the major role played by conformational disorder.

To investigate this question and more generally propose a strategy to study transient biomolecular interactions between flexible partners, we developed an approach combining NMR spectroscopy and corse grain modeling, where the information obtained on the two partners is used to implement a system specific force-field encoding the accessible experimental knowledge on that system.

With this approach we depicted the interaction between Spy, a recently discovered chaperone, and Im7, an in vivo substrate. The approach provided us a detailed picture at the residue level of a chaperone-substrate interaction allowing for a detailed description of the role of conformational disorder in chaperone action. The results were in agreement with multiple other experimental information including from x-ray crystallography, attesting the robustness of the method.

References:
Salmon, Ahlstrom et al. J. Am. Chem. Soc., 138, 9826–9839 (2016)
Horowitz, Salmon, Koldewey et al. Nat. Struct. Mol. Biol., 23, 691–697 (2016)
Salmon et al. Meth. Mol. Biol., vol 1764 (2018)

Speaker: Dr Loic Salmon (Centre de RMN à Très Hauts Champs (CNRS/ENSL/UCBL))
• 11:55
Atomic Insight into the Function and Activity of Molecular Chaperones 35m

Molecular chaperones are necessary for maintaining a functional proteome in the cell by preventing the aggregation of unfolded proteins and/or assisting with their folding. Despite the central importance of the binding of chaperones to unfolded substrates, the structural basis of their interaction remains poorly understood. The scarcity of structural data on complexes between chaperones and unfolded client proteins is primarily due to technical challenges originating in the dynamic nature of these complexes.

I will discuss how NMR spectroscopy can be used as an extremely powerful tool to determine the structural and dynamic basis for the recognition and interaction of unfolded proteins by molecular chaperones.

Speaker: Dr Charalampos Kalodimos (St Jude Children's Research Hospital)
• 10:30 12:30
EPR: Session 22 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 10:30
ESR microfluidics with picoliter samples 35m

Microfluidics is a well-established technique to process, synthesize and analyse small amounts of materials for chemical, biological, medical, and environmental applications. Typically, it involve the use of reagents with volume that is smaller than ~1 microliter – ideally even nano- or pico-liter. Conventional electron spin resonance (ESR), is typically carried out with ~ 1 ml of sample, thereby making it incompatible with most microfluidics applications. Here we show that by using a new class of miniature surface resonators, combined with photolithography to prepare microfluidics patterns, ESR can be applied to measure small samples, down to picoliter volume, without scarifying concentration sensitivity. Our experiments with resonators having mode volume of ~ 1 nano-liter can obtain good signal from solutions with ~ 1 mM spin concentration ,while smaller resonators can be used to measure even smaller volumes, but for higher spin concentrations. All our experiments are performed at room temperature, making our technique compatible with future microfluidics applications that would employ compact resonators, microfluidic chips, miniature magnet and a compact ESR-on a chip spectrometer. This could result in a complete new approach to process and measure paramagnetic liquid samples, applicable for a verity of chemical, biological, medical and environmental applications.

Speaker: Prof. Aharon Blank (Technion - Israel Institute of Technology)
• 11:05
Spin-labeled nanobodies as proteins’ conformational reporters towards in-cell EPR applications. 25m

Nanobodies (i.e. single-domain antibodies) are promising new tools for in-cell applications due to their low molecular weight, protein- and state- specificity, nano- or sub-nano-molar affinity to their target and the possibility to be inserted into cells. We propose here the use of spin-labeled nanobodies as conformational reporters of wild type unlabeled proteins via DEER spectroscopy.
We focused on a set of spin-labeled nanobodies targeting ABC transporters, proteins that couple the energy deriving from the binding and hydrolysis of ATP to large conformational changes that enable substrate translocation across membranes. These molecular machines have been studied in different environments (as detergent, liposomes and nanodiscs) via different techniques[1,2]; in this framework, the challenge ahead is the investigation of their conformational plasticity in a native environment.
We show here[3] the applicability of the use of gadolinium-labeled nanobodies against the heterodimeric exporter TM287/288: thanks to a “cocktail” of state- and non-state-specific nanobodies binding to different sites of the protein, we were able to obtain a fingerpint distance of the outward-facing state of the transporter and follow the conformational cycle of the unlabeled wild type protein. Orthogonal labels attached to the transporter were also used to corroborate and strengthen the findings. With this proof of principle, we then used a non-state-specific nanobody showing high affinity for both nucleotide binding domains of the homodimeric exporter MsbA to gather structural information in detergent, proteoliposomes, nanodiscs and inside-out vesicles. Our results show a strong structural dependency on the environment, proving the need of an investigation in a native environment. Reliable structural information at low micromolar protein concentrations were obtained, paving the way for the use of biocompatible Gd-labeled nanobodies in cells.

[1] Mi, W. et al., 2017, Nature, 549, 233-237.
[2] Borbat, P.P. et al., 2007, PloS biology, 5, e271.
[3] Galazzo, L. et al., 2019, in preparation.

Speaker: Dr Laura Galazzo (Ruhr University Bochum)
• 11:30
Quantitative sub-micromolar pulse dipolar EPR spectroscopy evidences high copper(II) labeling efficiency for double-histidine motifs 25m

Electron paramagnetic resonance (EPR) distance measurements provide highly accurate and precise geometric constraints. These have made valuable contributions to studies of the structures and conformations of biomolecules. Recently, application of double-histidine (dHis) motifs, coupled with CuII spin-labels has shown promise in even higher precision distance measurements.1 However, the non-covalent CuII-coordination approach is vulnerable to low binding-affinity. Earlier estimations of dissociation constants (KD) revealed micromolar to low millimolar KDs under EPR distance measurmeent conditions.1 As many challenging biomolecular targets are only stable at or below low micromolar concentration higher KDs are likely to limit the usefulness of this approach.

We have investigated the binding affinity directly from primary pulse dipolar (PD) EPR data.2 By combining spectroscopically orthogonal CuII and nitroxide spin-labels and performing RIDME (relaxation-induced dipolar modulation enhancement) distance measurements the uncertainty from speciation of the CuII spin-label is largely mitigated. By exploiting the superb sensitivity of this experiment and label combination we have demonstrated significant loading of dHis sites at submicromolar concentrations. This study demonstrates that the affinity for CuII-chelators is not limiting for PDEPR studies in the low micromolar range and that the combination of RIDME and the orthogonal spin-labels CuII and nitroxide makes submicromolar PDEPR experiments feasible.3

1. (a) T.F. Cunningham, M.R. Putterman, A. Desai, W.S. Horne, S. Saxena, ACIE 2015, 54, 6330; (b) S. Ghosh, M.J. Lawless, G.S. Rule, S. Saxena, JMR 2018, 286, 163.
2. (a) K. Ackermann, A. Giannoulis, D.B. Cordes, A.M.Z. Slawin, B.E. Bode, ChemComm 2015, 51, 5257; (b) A. Giannoulis, M. Oranges, B.E. Bode, ChemPhysChem 2017, 18, 2318; (c) A. Giannoulis, K. Ackermann, P. Spindler, C. Higgins, D.B. Cordes, A.M.Z. Slawin, T.F. Prisner, B.E. Bode, PCCP, 2018, 20, 11196.
3. J.L. Wort, K. Ackermann, A. Giannoulis, A.J. Stewart, D.G. Norman, B.E. Bode, under review.
Speaker: Dr Bela Bode (University of St Andrews)
• 11:55
High-Frequency Electron-Nuclear Double Resonance to Study Biomolecules 35m

Electron-nuclear double resonance (ENDOR) and dynamic nuclear polarization (DNP) are two techniques based on polarization transfer between electron and nuclear spins. Despite differences in the experimental realization, their similarities rely on the detailed mechanism of hyperfine interactions. The lecture will give an overview of our recent developments in these two methods in solids (ENDOR) and solution (Overhauser DNP) to study biological systems. To this end, design and implementation of coupled EPR/NMR experiments at various microwave frequencies, particularly in the high-frequency/high-field EPR regime, has been in focus.
We have recently implemented a spectrometer to perform routine ENDOR spectroscopy at 263 GHz/9.4 Tesla. Spectrometer design, performance as well as the demonstration of unprecedented spectral resolution in ENDOR for studies of protein radicals is presented. Moreover, we illustrate that high frequency ENDOR in combination with a new 19F/nitroxide labelling strategy can be used to measure interspin distances in the range 0.5 – 1.5 nm. Finally, expansion of 13C dynamic nuclear polarization in the liquid state towards high magnetic fields is discussed. We could recently measure 13C NMR signal enhancements on small molecules up to fields of 9.4 Tesla.

Speaker: Prof. Marina Bennati (MPI for Biophysical Chemistry & University of Göttingen)
• 10:30 12:30
Instrumentation: Session 25 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 10:30
Wither the Spin Diffusion Barrier 35m

In the last few years microwave driven dynamic nuclear polarization (DNP) has become the method of choice to enhance signal intensities in a variety magic angle spinning (MAS) NMR experiments. In particular, because of the large signal enhancements, it is used to address a variety of important chemical, biological and physical questions that are otherwise inaccessible. Despite the success of DNP, there is not a detailed understanding of the manner in which the high polarization of the electron is transferred to the surrounding nuclei or where these nuclei are located relative to the polarizing agent in an amorphous glassy matrix. In a number of different papers the size of the “spin diffusion barrier” surrounding the paramagnetic center has been postulated to be as large 20-40 Å, and therefore to encompass a large number of spins. In this paper we perform an experimental and theoretical analysis of the recently rediscovered three-spin solid effect (TSSE) and show that it is exquisitely sensitive to the electron-nuclear distances. We exploit this feature to determine the size of the spin diffusion barrier surrounding the trityl radical in a glassy glycerol–water matrix and therefore the location of the protons involved in the initial transfer step. They are less than 6 Å from the electron and located on the perimeter of the trityl molecule. Thus, there is effectively not a spin diffusion barrier in the case of trityl in DNP juice. Furthermore, 1H ENDOR experiments indicate that the electron polarization is first transferred to the intramolecular 1H’s on the trityl radical, and then via spin diffusion to glycerol molecules in intimate contact with the trityl and subsequently to other 1H’s in the solvent.

Speaker: Prof. Robert G. Griffin (MIT)
• 11:05
Squeeze It Until It Breaks: In-situ NMR at Geophysically Relevant Conditions 25m

Recent developments in magnetic flux tailoring techniques paved the way for NMR in high pressure diamond anvil cells at multi-megabar pressures. Using a combination of physical vapor deposition and focused ion beam milling techniques, NMR resonator structures based on the principles of recently developed Lenz lenses can be realized with 1 µm spatial resolution. These structures have been demonstrated to combine the mechanical robustness and spin sensitivity required for geophysically extreme conditions, i.e. pressures well above 100 GPa and temperatures above 1000 K.
Here we present our work of the last 3 years at the Bavarian Geoinstitute which led to this major technological advancement.

Speaker: Dr Thomas Meier (Bavarian Geoinstitute)
• 11:30
Microscale NMR-spectroscopy with femtomole sensitivity using diamond quantum sensors 25m

Nuclear magnetic resonance (NMR), one of the most powerful analytical techniques in chemistry and life science, is typically limited to macroscopic volumes due to its inherent low sensitivity. This excludes NMR spectroscopy from analysis of microscopic samples sizes such as in single-cell biology or in microfluidic applications. In recent years, it has been shown that NMR signals can be detected from nano- to microscale volumes by a new sensor class – quantum sensors based on defects in the diamond lattice - the nitrogen-vacancy (NV) center. However, these experiments were limited by a low spectral resolution and to pure samples with high viscosity, which precludes practical applications in chemistry. Here, I will present our recent results where we could overcome these basic problems. First, I will describe how NV-centers can be used to detect NMR signals from picoliter sample volumes on the surface of the diamond chip with high spectral resolution (~1 Hz). Second, I will discuss our newest results on improving the molecular sensitivity of this approach by hyperpolarizing nuclear sample spins. This technique combines microscopic-scale NV-NMR with a fully integrated Overhauser dynamic nuclear polarization scheme which reaches femtomole sensitivity. I will provide an overview of this rapidly developing technology and discuss potential applications, such as single cell metabolomics.

Speaker: Dr Dominik Bucher (Technical University of Munich)
• 11:55
Solid State NMR Probes for 1.5 GHz Spectrometer 35m

We report on design of solid-state NMR probes for the 1.5 GHz NMR magnet at NHMFL facility in Florida.1 This magnet is open to external NMR users, offering sensitivity and resolution enhancements, and the new opportunities in NMR of quadrupolar and low-γ nuclei. We will discuss strategy for making higher-field solid-state NMR probes for materials and biological applications, while preserving the large sample volumes that NMR community grew accustomed to in the sub-GHz NMR range. We constructed multi-resonant direct-detection NMR probes, MAS and static, with sample sizes in 2, 3, 4, 5 mm range. These probes are part of the 1.5 GHz Bruker NEO spectrometer. We will report performance and NMR spectra.

Both 1HXY and 1HX probes use Low-E coils designs that separate high- and low-frequency RF circuits2. This allows efficient direct detection at higher 1H frequencies with far less trade-off in sample size or sensitivity. The triple-resonance 1HXY circuit is arranged on tune cards1 for quick interchange of X/Y isotopes. The static probe has modular slide-in sample coils that accommodate different sample shapes and sizes: 3, 4, 5 mm round and 4x4 mm square profiles.

Each probe required additional hardware to perform NMR in the hybrid magnet consisting of series-connected superconducting coil and resistive DC insert.1 Active field-regulation compensates B0 fluctuations, enabling signal averaging and 2D NMR experiments. An inductive sensor tracks B0 field and drives an outer correction coil to cancel fast 60 Hz fluctuation and harmonics. An external 7Li NMR field lock is incorporated to correct slower B0 drift. A MnCl2-doped LiCl solution serves as the lock sample. Spatial B0 inhomogeneity of < 0.9 ppm over 1cm3 DSV is achieved by combining active shims with the passive ferroshims in the probehead.

1Gan Z. et al., J.Magn.Reson. 2017; 284, 125.
2Gor’kov P. et al., J.Magn.Reson. 2007; 185, 77.

Speaker: Prof. Peter Gor'kov (National High Magnetic Field Laboratory)
• 10:30 12:30
Small Molecules: Session 21 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 10:30
Boosting the NMR characterization of small- to medium-sized molecules 35m

The recently introduced CLIP-COSY[1] experiment providing homonuclear correlation spectrum with high quality clean in-phase multiplets expedites the assignment of scalar coupled proton spin network, aiding the structure elucidation of small- and medium-sized molecules. The resolution of COSY spectra is, however, limited by the inherently small chemical shift dispersion of proton resonances.

Last year we devised HSQC-variant of the CLIP-COSY experiment[2] for enhancing the resolving power of the method by utilizing the increased chemical shift range of heteronuclei. Herein, it will be demonstrated that the performance of the original HSQC-CLIP-COSY experiment can be further boosted with incorporation of heteronuclear spin echo block(s) in the pulse sequence, allowing phase-editing of crosspeaks of different types. The edited HSQC-CLIP-COSY experiments providing well-resolved spectra have great promise, enabling straightforward NMR assignments for carbohydrates and peptides and for other types of molecules.

The applicability of the CLIP-COSY approach can be extended for binding studies using a combined STD-CLIP-COSY(relayed) experiment. The resulting well-resolved, high quality 2D spectrum makes feasible to separate overlapping signals of 1D STD, allowing quantitative assessment of binding.

In small-molecule NMR there has always been a need for an experiment to distinguish between two- and three-bond correlations for quaternary 13C-s in a sensitive and reliable way. Last year we introduced a simple, non-selective experiment, SEA XLOC[3], which is capable to distinguish these correlations in a novel way and is applicable for all 13C multiplicities.

In the talk the scope and limitations of these experiments will be illustrated by applications to spins systems of varying complexity.

1. Koos, MRM, Kummerlöwe, G, Kaltschnee, L, Thiele, CM, Luy, B,
Angew. Chem., Int. Ed., 55, 7655-7659, 2016
2. Gyöngyösi, T, Timári, I, Haller, J, Koos, MRM, Luy, B, Kövér, KE, ChemPlusChem, 83, 53-60, 2018
3. Gyöngyösi, T, Nagy TM, Kövér, KE, Sørensen, OW,
Chem. Commun., 54, 9781-9784, 2018
Speaker: Prof. Katalin E. Kövér (University of Debrecen)
• 11:05
Prebiotic Organization of Biomolecules on Mineral surfaces 25m

The structural organization of the interface characterizing binding, assembly and recognition of biomolecules on inorganic surfaces has attracted considerable attention in the domains of catalysis and prebiotic chemistry. In the context of origins-of-life chemistry, this study is focused on the catalytic effect of a silica surface on amino acids condensation, more precisely on two amino acids, Leucine and Glutamic acid, adsorbed on silica nanoparticles. It takes into consideration the important effect of hydration on the system. Solid state NMR is a choice technique for surface characterization and observation of bonding at the atomic level, as well as local proximities between the amino acid and the silica surface sites.
13C and 15N CP-MAS NMR and 2D-HETCOR experiments allowed to characterize the structure of adsorption complexes at the interface, involving amino acids, surface groups, and water molecules. At high surface coverages, both crystalline and adsorbed Leucine exist in the samples, while only adsorbed forms of leucine were observed at rather low surface coverage (3%Leu/SiO2). The same type of approach was applied to Glutamic Acid, but the adsorbed form was predominant only at much lesser loadings (0.3%Glu/SiO2) than those observed for Leucine. The sensitivity of NMR compared to other characterization techniques is low; however, we were able to use 13C and 15N-enriched amino acids which gave us information on the evolution of chemical shifts, after adsorption and upon drying in careful conditions, even for such low loadings.
Hydration was a key parameter in our experiments and samples with varying degrees of hydration were studied experimentally by NMR, and also by computational methods such as DFT simulation. Water turned out to mediate the interaction of molecules with silanols of the silica surface.

Speaker: Mr Hagop Abadian
• 11:30
Recent advances in polypeptidic thermoresponsive alignment media for organic compounds 25m

Anisotropic NMR parameters become increasingly important in organic structure elucidation for the determination of conformations and relative configuration of natural products, synthesized compounds and catalysts.[1]
For anisotropic NMR parameters to be obtained suitable alignment media are necessary. The use of lyotropic liquid crystals from helically chiral polymers is especially intriguing in that respect as they additionally allow for enantiodiscrimination.[2]
We have recently synthesized several homopolypepides[3], which form lyotropic liquid crystals, are suitable for the measurement of anisotropic NMR observables, show excellent enantiodiscrimination and furthermore induce different orientations at different temperatures. They are thus considered thermoresponsive.
The possibility to induce different orientation at different temperatures thus alleviates the need to use more than one alignment medium in cases of ambiguity.
The intriguing properties of these new thermoresponsive alignment media will be described in this presentation.

References:
[1] For reviews see: : C. M. Thiele, Eur. J. Org. Chem. 2008, 5673-5685; V. Schmidts, Magn. Reson. Chem. 2017, 55, 54-60.
[2] For review see: P. Lesot, J. Courtieu, Prog. Nucl. Magn. Reson. Spectrosc. 2009, 55, 128-159.
[3] M. Schwab, D. Herold, C. M. Thiele, Chem. Eur. J. 2017, 23, 14576-14584; M. Schwab, V. Schmidts, C. M. Thiele, Chem. Eur. J. 2018, 24, 14373-14377; S. Jeziorowski, C. M. Thiele, Chem. Eur. J. 2018, 24, 15631-15637.

Speaker: Prof. Christina M. Thiele (Technische Universität Darmstadt)
• 11:55
Fast quantitative 2D NMR for metabolomics 35m

NMR is a major tool in metabolomics thanks to its non-destructive and highly reproducible character. NMR metabolomics include untargeted analysis where spectral fingerprints are analyzed with statistical tools to highlight potential biomarkers, and targeted methods which aim at accurately quantifying multiple metabolites. Most studies rely on 1D NMR which suffers from ubiquitous spectral overlap that hampers the accurate determination or quantification of biomarkers.

In this lecture, we will try to answer the following question: can we replace 1D by 2D spectroscopy in NMR metabolomics? We illustrate, through several examples, how 2D NMR can advantageously replace 1D spectra in both targeted and untargeted workflows, provided that fast and reproducible methods are employed to fit the high-throughput requirements of metabolomics [1].

For untargeted methods, a variety of accelerated pulse sequences can be used, such as those relying on ultrafast 2D NMR. These 2D methods lead, after statistical analysis, to a better determination of biomarkers, as we recently showed on a food chemical safety example [2]. When peak overlap is critical –for instance in spectra recorded on a benchtop spectrometer– 2D NMR can even yield a better separation between sample groups [3].

In the case of targeted methods, tailored solutions are needed so that 2D NMR can be used for the simultaneous quantification of multiple analytes in complex samples. We will describe different strategies –all including accelerated acquisition methods– to quantify metabolites from 2D NMR spectra, either relying on analytical chemistry approaches [4] or on the design of “intrinsically quantitative” 2D experiments [5].

[1] J. Marchand, et al., Curr. Op. Biotechnol. 2017, 43, 49-55.
[2] J. Marchand, et al., Metabolomics 2018, 14, 60.
[3] B. Gouilleux, et al., Food Chemistry 2018, 244, 153-158.
[4] T. Jézéquel, et al., Metabolomics 2015, 11, 1231-1242.
[5] J. Farjon, et al., Anal. Chem. 2018, 90, 1845-1851.

Speaker: Prof. Patrick Giraudeau (Université de Nantes)
• 12:30 13:30
Brunch 1h
• 13:30 14:20
Prize Lectures: GDCh Prize Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 13:35
Complex Formation of the Tetracycline‐Binding Aptamer Investigated by Specific Cross‐Relaxation under DNP 10m

Specific Cross-Relaxation Enhancement by Active Motions under DNP (SCREAM-DNP) is a method which relies on direct polarization transfer in solid-state DNP at typical DNP temperatures.[1] The mechanism is based on cross relaxation between 1H and 13C. It is generated by the internal dynamics of methyl groups resulting in negative enhancement and inverted 13C MAS NMR signal in a direct DNP experiment.[2] Furthermore, this effect can be suppressed by 1H saturation. Through mathematical subtraction, we can exclusively observe magnetization which was generated by cross relaxation. Therefore, the use of methyl groups as a specific promotor for polarization transfer opens new applications in DNP.
In this work, we show the application of SCREAM-DNP on a tetracycline-binding RNA aptamer. Here, CH3 groups were introduced into the biomolecular complex by non-covalent interaction between the aptamer and its highly specific ligand. Thereby, we use tetracycline which carries three CH3-groups as a source of cross-relaxation enhancement for complex formation studies. Moreover, we can influence the reorientation dynamics of methyl groups to a significant degree by changing the temperature, resulting in an increase of cross-relaxation efficiency. In conclusion, SCREAM-DNP is a promising method for different applications, especially in site-specific DNP-studies.
[1] Aladin et al., Angew. Chem. Int. Ed. 2019, 58, 4863.
[2] Daube et al., J. Am. Chem. Soc. 2016, 138, 16572.

Speaker: Victoria Aladin (Goethe University Frankfurt)
• 13:45
Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR 10m

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized compound to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.

Speaker: Daniel Friedrich (Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany)
• 13:55
Time evolution of coupled spin systems in a generalized Wigner representation 10m

So-called phase-space representations such as Wigner functions, are a powerful tool for representing quantum states and characterizing their time evolution in the case of infinite-dimensional quantum systems and have been widely used in quantum optics and beyond. Continuous phase spaces have also been studied for the finite-dimensional quantum systems of individual spins. However, much less was known for coupled spin systems, and we present a complete theory of Wigner functions for this case. In particular, we provide a self-contained Wigner formalism for describing and predicting the time evolution of coupled spins which lends itself to visualizing the high-dimensional state space in a structured and intuitive way. We completely treat the case of an arbitrary number of coupled spins 1/2, thereby establishing the equation of motion using Wigner functions. The explicit form of the time evolution is then calculated for up to three spins 1/2. The underlying physical principles of our Wigner representations for coupled spin systems are illustrated for several NMR examples. This talk is based on Annals of Physics DOI 10.1016/j.aop.2018.11.020 (in press).

Speaker: Dr Bálint Koczor (Technische Universität München, now University of Oxford)
• 14:05
Dynamic nuclear polarization of 13C in the liquid state over a 10 Tesla field range 10m

DNP in liquids is driven by electron-nuclear cross-relaxation, known as Overhauser effect (O-DNP). When relaxation is dominated by scalar hyperfine interaction, the enhancements can reach three orders of magnitudes, as recently reported for $^{13}$C-DNP at 3.4 T [1].
Hereby we present a systematic study performed at different magnetic fields on model systems doped with nitroxide radical (TEMPONE) as polarizing agent [2]. $^{13}$C signal enhancements on organic small molecules in liquids at room temperature were observed as high as 800 at 1.2 Tesla and 600 at 9.4 Tesla. An accurate determination of Overhauser parameters allowed us to disclose the primary role of the scalar hyperfine interaction to the $^{13}$C nuclei as mediated by molecular collisions in the sub-picoseconds timescale.
Experimental measurements performed at 1.2 T, 9.4 T, and 14 Tesla allowed us to complete the characterization of the polarization transfer efficiency over a broad frequency range and described it by the subtle combination of dipolar and scalar relaxation.
Furthermore, we recognized that a proper choice of polarizing agent/target system is essential to optimize the efficiency of scalar O-DNP. Indeed, fullerene-nitroxide derivatives are superior to TEMPONE radical as polarizing agent at low fields, while halogens atoms (Cl, Br) bound to the target C nucleus seems to favor the scalar interaction.
The observation of sizable DNP of $^{13}$CH$_2$ and $^{13}$CH$_3$ groups in organic molecules at 9.4 T preserving NMR resolution opens perspectives for a broader application of this method as a tool to address $^{13}$C-NMR sensitivity issues at high fields.

[1] Liu G., Levien M., Karschin N., Parigi G., Luchinat C., and Bennati M. Nat. Chem. 9, 676-680 (2017)
[2] Orlando T., Dervisoglu R., Levien M., Tkach I., Prisner T.F., Andreas L.B., Denysenkov V., Bennati M. Angew. Chem. Int. Ed. 58, 1402-1406 (2019)

Speaker: Dr Tomas Orlando (RG ESR Spectroscopy, Max Planck Institute for Biophysical Chemistry)
• 14:20 15:00
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 14:20
Stronger Together: Bacterial Weaving of Functional Amyloid and Polysaccharide Composites to Assemble Multicellular Biofilm Communities 40m

Biofilms are implicated in serious infectious diseases and have emerged as a target for anti-infectives. Our research program is inspired by the challenge and importance of elucidating chemical structure and function in complex biological systems and we strive to transform our discoveries into new therapeutic strategies. We have introduced new approaches integrating solid-state NMR with microscopy and biochemical and biophysical tools to reveal how amyloid-associated biofilm assembly and architecture in E. coli is influenced by chemical and molecular composition. Solid-state NMR is serving as a powerful discovery tool in these efforts and we recently reported the unprecedented discovery of a naturally produced chemically modified cellulose produced by E. coli and Salmonella strains. Solid-state NMR of the intact cellulosic material enabled the elucidation of the zwitterionic phosphoethanolamine modification and we identified the genetic and molecular basis for its assembly. I will report on our efforts to understand and describe the macromolecular assembly of the amyloid-polysaccharide composites in E. coli and their relation to biofilm function.

Speaker: Prof. Lynette Cegelski (Stanford University)
• 15:00 18:00
Tutorials Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 15:00
Basics of MRI 1h
Speaker: Bernd Ittermann (Physikalisch-Technische Bundesanstalt (PTB))
• 16:00
Quantum-Chemical Methods 1h
Speaker: Prof. Martin Kaupp (Technische Universität Berlin)
• 17:00
You Spin Me Right 'Round: Tensors and Rotations in NMR 1h
Speaker: Prof. Leonard Mueller (UC Riverside)
• 15:15 16:15
Society Meetings: GDCh Members' Meeting Lecture Hall C

Lecture Hall C

Henry Ford Building

• 18:10 19:40
Music 1h 30m Lecture Hall A

Lecture Hall A

Henry Ford Building

The EUROISMAR Friends, its Chamber Orchestra and Duo Dopico with Friend
Ville Telkki and Anu Kantola (songs by Schubert and Sjoberg)
Li Zhao (Chinese piano song)
Norbert Lutz (clarinet improvisations)
Jordan Chill (Beethoven sonata and improvisation)
Oleg Anztukin, solo guitar
Rob Tycko, Henrike Heise, and Zoltan Szakacs (Suite by Milhaud)

INTERMISSION

Rob Tycko, Henrike Heise, Duo Dopico and Friend (Mozart quintet)

• Thursday, 29 August
• 08:40 10:10
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 08:40
Optically-pumped dynamic nuclear polarization under ambient conditions via nitrogen-vacancy centers in diamond 40m

A broad effort is underway to improve the sensitivity of nuclear magnetic resonance through the use of dynamic nuclear polarization (DNP). Nitrogen-vacancy (NV) centers in diamond offer an appealing platform because these paramagnetic defects show efficient optical pumping at room temperature. This presentation focuses on the spin dynamics of NVs coupled to substitutional nitrogen (the so called P1 center) as a platform for DNP, with emphasis on recent schemes designed for powder geometries and optimal polarization transfer across the diamond surface. I will also discuss new phenomenology under NV-P1 cross-relaxation conditions revealing record fast nuclear spin diffusion constants as well as quick, homogeneous thermalization between bulk and strongly hyperfine-coupled nuclei. These observations highlight the need for DNP descriptions beyond classical models based on spin diffusion barriers.

Speaker: Prof. Carlos Meriles (CUNY - CIty College of New York)
• 09:30
High-resolution NMR spectroscopy applied for field inhomogeneity and spectral congestion 40m

NMR spectroscopy presents a non-invasive detection technique for molecular structure elucidation and dynamic effect analyses. In general NMR applications, spectral resolution is the key index determining the availability of resulting spectra. Due to limited chemical shift ranges and appended J coupling splittings, conventional 1H NMR spectra are subject to spectral congestions in complex samples. In addition, there exist adverse experimental conditions in which magnetic fields suffer from spatial or temporal inhomogeneity, constituting the second factor degrading spectral resolution in 1H NMR applications. Therefore, an NMR method available for high-resolution NMR measurements under the condition of inhomogeneous fields and complex samples is greatly demanded.
In our previous studies, a series of NMR methods based on intermolecular multiple-quantum coherence (iMQC)[1] is proposed for high-resolution applications in inhomogeneous magnetic field conditions. And other pure-shift based methods are also designed to eliminate J coupling splittings and further enhance spectral resolution.[3] For example, a high-resolution NMR method, named UPSIF, has been propsed to extract high-resolution 1D pure shift or 2D J-resolved spectra, suitable for direct analyses on biological samples. The proposed method is designed based on the combination of constant-time module and iMQC scheme. The constant-time module constitutes a direct decoupling manner for removing J couplings and extracting pure chemical shifts. The iMQC scheme is proved to be immune to field inhomogeneity. The performance of the proposed high-resolution method is demonstrated by experiments on biological samples with intrinsic field inhomogeneities and in situ electrochemical detection under externally adverse field conditions. Our proposed methods can be applied for high-resolution measurements under the condition of field inhomogeneity and spectral congestion.

Reference
[2] Z. Chen, S.H. Cai, Y.Q. Huang, Y.L. Lin, Prog. Nucl. Magn. Reson. Spectrosc., 90-91, 1-31, 2015.
[3] K. Zangger, H. Sterk, J. Magn. Reson., 124, 486−489, 1997.

Speakers: Prof. Zhong Chen (Xiamen University), Prof. Yuqing Huang (Department of Electronic Science, Xiamen University, Xiamen, China)
• 09:20 09:30
Society Meetings: ISMAR General Assembly Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 10:10 10:30
Coffee 20m
• 10:30 13:00
Biomolecules: Session 28 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 10:30
Spinning faster: Developments and Applications to Biological NMR 35m

Solid-state Protein NMR: Resolution and Sensitivity for Fast MAS Experiments
Beat H. Meier1, Alexander Malär1, Maarten Schledorn1, Anahit Torosyan1, Susanne Penzel, Thomas Wiegand1, Denis Lacabanne2, Albert A. Smith1, Nils-Alexander Lakomek1, Alons Lends, Vlastimil Jirasko, Lauriane Lecoq2, Matthias Ernst1, Anja Böckmann2
1Laboratory of Physical Chemistry, ETH Zürich, Vladimir-Prelog-Weg 2, 8903, Zurich, Switzerland.2Institut de Biologie et Chimie des Protéines, Bases Moléculaires et Structurales des Systèmes Infectieux, Labex Ecofect, UMR 5086 CNRS, Université de Lyon, 7 passage du Vercors, 69367 Lyon, France

Fast magic-angle spinning allows for highly resolved protein proton spectra and gives access to site-specific relaxation data in solid proteins. Fast MAS requires small rotor diameters and correspondingly small sample amounts in the order of 100 picoliters, limiting the signal-to-noise ratio. The limitations encountered depend on the linewidths and coherence lifetimes and will be discussed in detail.
Spectra at 150 kHz MAS (and hopefully higher) will be discussed and compared to spectra around 100 kHz.
Applications presented include dynamical aspects of the helicase DnaB, a bacterial, ATP-driven enzyme that unwinds double-stranded DNA during DNA replication. Conformations mimicking the pre-hydrolytic state, the transition state and a post-hydrolytic state are arrested and then investigated by 3D NMR spectroscopy. In addition, spectra of viral capsids, membrane proteins, and of the Rpo4/7 protein complex will be discussed.

Speaker: Prof. Beat H Meier (ETH Zurich)
• 11:05
Sensitivity-Enhanced Protein Solid-state NMR using Ultra-fast MAS and Structural Studies of Alzheimer’s Amyloid-β 25m

This work involves two separate topics on our ongoing progress of protein SSNMR methods using ultra-fast MAS and solid-state NMR (SSNMR) applications to amyloid proteins. First, we discuss resolution and sensitivity enhancement in 1H-detected biomolecular SSNMR under ultra-fast magic angle spinning (UFMAS) conditions (≥ 80 kHz) in a high magnetic field (1H frequency: 750-900 MHz).1,2 Our data on protein microcrystal GB1 and amyloid-β (Aβ) fibril show that traditionally time-consuming 3-5D biomolecular SSNMR is feasible for signal assignments and structural examination of proteins in a nano-mole-scale with this approach. Our discussion will include drastic sensitivity enhancement by novel polarization-transfer schemes and other methods for multi-dimensional SSNMR using ultra-fast MAS. We briefly introduce our nation-wide effort to construct a 1.3 GHz NMR at RIKEN.

Second, we examine structures, kinetics, and functions of amyloid-β using solid-state NMR (SSNMR). Increasing evidence suggests that formation and propagation of misfolded aggregates of 42-residue Aβ42, rather than the more abundant 40-residue Aβ40, provokes the Alzheimer’s cascade. Our group recently presented the first detailed atomic model of Aβ42 amyloid fibril based on SSNMR data.3 The result revealed a unique structure that was not previously identified for Aβ40 fibril. Based on the results and additional SSNMR data, we discuss how amyloid fibril structures affect “prion-like” propagation across different Aβ isoforms, including WT Aβ40 and E22G pathogenic mutant of Aβ40.4 We also discuss SSNMR-based structural analysis of toxic spherical assembly of Aβ, including one that was identified from brains affected by AD.5 The results provide insight into amyloid misfolding of Aβ42 in Alzheimer’s disease.

(1) Wickramasinghe, N.et al. Nat. Methods 2009, 6, 215.
(2) Ishii, Y.et al. J. Magn. Reson. 2018, 286, 99.
(3) Xiao, Y.et al. Nat. Struct. Mol. Biol. 2015, 22, 499.
(4) Yoo, B.et al. JACS 2018, 140, 2781.
(5) Parthasarathy, S.et al. JACS 2015, 137, 6480.

Speaker: Dr Yoshitaka Ishii (Tokyo Institute of Technology)
• 11:30
Atomic resolution characterization of a folding intermediate by pressure-jump NMR 25m

Understanding how proteins fold into stable structures without external assistance remains one of the major open questions in biophysics. The ability of NMR to report atomic resolution structural information for both folded and unfolded proteins makes it a uniquely powerful to study protein folding. However, typical experiment times of hours to days are incompatible with the typical microsecond to second timescale of folding. Here, we use a new hardware which rapidly and repeatedly switches sample pressure from native (1 bar) to unfolded (2.5 kbar) conditions in less than 3 ms to study the folding of a pressure-sensitized mutant of Ubiquitin. Recently, we demonstrated the presence of two parallel folding pathways where one involves a meta-stable intermediate. Initially, we developed a set of experiments to obtain site-specific 15N and 13C’ chemical shifts of this intermediate state using stroboscopic chemical shift measurement or a reverse-sampled indirect evolution period. In addition, as in this case the intermediate has a relatively long lifetime, we have modified standard 3D experiments to measure the first chemical shift evolution (1H, 15N, 13Ca, 13C’) ~60 ms after the pressure drop, which allows direct observation of cross peaks to intermediate state frequencies. Complemented with other NMR observables such as pressure-jump NOEs and RDCs, we derived a structural model for the intermediate. The model reveals a structure that is similar to the native state of wild type Ubiquitin but but contains multiple non-native H-bonds. This method opens the way for the structural characterization of folding intermediates at atomic resolution.

Speaker: Dr Cyril Charlier (NIH)
• 11:55
Monitoring phosphorylation events at the interface between the nuclear envelope and chromatin 35m

The molecular mechanisms that regulate genome organization in the mammalian interphase nucleus are largely unclear. At the interface between the nuclear membrane and chromatin, the inner nuclear envelope contains both nucleoskeleton filaments (lamins) and transmembrane proteins (NETs). Lamins tether heterochromatin to the nuclear envelope and modulate chromosome territory positions. Tissue specific expression of NETs also influences genome organization. Phosphorylation regulates localization and interactions of the nuclear envelope proteins during cell cycle and after a mechanical stress. We focused on a complex formed by lamin A/C, emerin (one of the best characterized NETs) and the chromatin binding protein BAF. These proteins contain intrinsically disordered regions (IDRs) that are highly phosphorylated in cells. We showed that BAF dimer mediates the interaction between lamin A/C and emerin, we solved the 3D structure of the complex (1), and we analysed the impact of cell cycle-dependent BAF phosphorylation by the kinase VRK1 on BAF structure and complex assembly. Emerin exhibits a large IDR that is responsible for self-assembly and binding to structural proteins (lamin, actin, tubulin) (2). We also described the impact of mechano-dependent emerin tyrosine phosphorylation by the Src kinase on emerin structure and binding properties. Finally, we identified defective phosphorylation and binding events associated to muscular dystrophy (3) and premature ageing syndromes (1).
References: (1) Samson et al., Nucleic Acids Res. 46, 10460-10473 (2018). (2) Samson et al., FEBS J. 284, 338-352 (2017). (3) Herrada et al., ACS Chem Biol. 10, 2733-2742 (2015).

Speaker: Dr Sophie Zinn-Justin (Institute for Integrative Biology of the Cell, CEA, CNRS, Université Paris-Saclay, CE-Saclay, Gif/Yvette, France)
• 12:30
Protein phase diagrams determined by high-pressure NMR 30m

The accessible free energy landscape is a generic property of proteins, which determines both their protein folding pathways and their biological function. This landscape can be explored by determining the thermodynamic stability of proteins at different pressures and temperatures. We combine these variations with NMR spectroscopy to gain molecular resolution. For two proteins (apoKti11 [1] and GB1 [2]) we could determine the pressure-temperature phase diagram, which allowed to explain the stabilization of the proteins at elevated pressures in thermodynamic (volume and entropy changes) and structural terms (conformation plasticity and pKa value changes). For apoKti11, for the first time we could disclose a hyperbolic pressure-temperature phase diagram. Pressure induced changes in the protein folding rates of GB1 derived from CPMG relaxation dispersion allocated the stabilization effect to the native state while the transition and unfolded states remained unaffected.

Speaker: Prof. Jochen Balbach (Martin-Luther-University Halle-Wittenberg)
• 10:30 13:00
Hyperpolarization in Materials: Session 26 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 10:30
Dynamic Phenomena in Fuel Cells and Batteries Investigated by Various NMR Techniques 35m

For mechanistic investigation of energy conversion/storage systems such as fuel cells and batteries, integral understanding of not only electrochemical phenomena but also chemical reactions and dynamics of chemical species in the systems is essential. We have employed various NMR techniques to reach the goal and some results will be presented.
The water and proton dynamics in Nafion polymer electrolyte membrane (PEM) will be introduced first. PEM is a main constituent, separating cathode and anode electrodes, in low temperature fuel cells such as hydrogen fuel cells and direct alcohol fuel cells (DAFC). For this study, Overhauser dynamic nuclear polarization as well as slow magic angle spinning was employed. The results indicated that hydrophobic surface enhances water dynamics. Multiscale water/proton dynamics in PEM will be discussed together with non-MR data in addition.
Secondly, the reaction mechanism of DAFC will be discussed in terms of cons and pros of toroid cavity detector and flow NMR techniques employed for this study.
Thirdly, the role of histidine additive in vanadium redox flow battery (VRFB) investigated with multinuclear solution NMR and first-principles calculations will be explained. The results showed that histidine additive dynamically interacts with vanadium ions forming an outer-sphere [VO2+(V)–histidine2+] complex. This dynamic complexation was found to prevent the aggregation of VO2+(V) ions and subsequent growth of V2O5, resulting in suppression of V2O5 precipitation. This leads to the improved stability and performance of VRFB.
Finally, challenges and promising aspects of MR techniques for investigation of dynamic phenomena occurring in electrochemical systems and their constituents will be summarized.

Speaker: Dr Oc Hee Han (Korea Basic Science Institute )
• 11:05
Bulk Hyperpolarization of Inorganic Materials 25m

We have recently shown how the bulk of proton-free inorganic solids can be hyperpolarized using dynamic nuclear polarization, resulting in sensitivity enhancements in MAS experiments.$^1$ This is achieved by hyperpolarizing nuclei near the particle surface with impregnation DNP and then allowing slow spontaneous spin diffusion between weakly magnetic nuclei to relay the hyperpolarization towards the bulk.
Pulse cooling is a version of this method that uses multiple contact cross-polarization for bulk hyperpolarization. We show how to maximize sensitivity gains in pulse cooling by optimization of the pulse parameters and delays. In addition, we show how to improve sensitivity by modulating the MAS rate during the experiment, which can provide gains of up to a factor 3.5 for the $^{119}$Sn spectra of SnO$_2$, compared to a constant MAS rate.$^2$ We also show how multidimensional experiments can be used to probe the pathway of spin diffusion, particularly for the spectra of compounds with more than one bulk chemical shifts.

1. Bjorgvinsdottir, S.; Walder, B. J.; Pinon, A. C.; Emsley, L., Bulk Nuclear Hyperpolarization of Inorganic Solids by Relay from the Surface. J Am Chem Soc 2018, 140 (25), 7946-7951.
2. Bjorgvinsdottir, S.; Walder, B. J.; Matthey, N.; Emsley, L., Maximizing nuclear hyperpolarization in pulse cooling under MAS. J Magn Reson 2019, 300, 142-148.
Speaker: Snaedis Björgvinsdóttir (EPFL)
• 11:30
Room-temperature triplet dynamic nuclear polarization in nanoporous materials and in water 25m

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) are powerful and versatile methods in modern chemistry and biology fields. Nevertheless, they suffer from intrinsically limited sensitivity due to the low nuclear spin polarization at ambient temperature. One of the promising methods to overcome this limitation is dynamic nuclear polarization (DNP) that transfers spin polarization from electrons to nuclei. In particular, DNP based on photo-excited triplet (triplet-DNP) is promising, since it allows the hyperpolarization at room temperature. In typical scheme of triplet-DNP, the spin-selective intersystem crossing (ISC) produces the large electron spin polarization in the excited triplet state sublevels, and this polarization is effectively transferred to nuclear spins by a pulsed microwave irradiation for satisfying Hartmann-Hahn condition, so-called integrated solid effect (ISE).
Previous studies of triplet-DNP have been limited to dense crystalline and amorphous materials, and it remains difficult to hyperpolarize biology-relevant probes. To overcome this limitation, we introduce the chemistry of metal-organic frameworks (MOFs) to the field of triplet-DNP (J. Am. Chem. Soc., 2018, 140, 15606). The nanoporous structure of MOFs allows the accommodation of polarizing agents as well as other guest molecules. This work paves the way towards the hyperpolarization of various probe molecules at room temperature for imaging applications.
Another important challenge of triplet-DNP is to develop air-stable polarizing agents. Since the first report of room-temperature triplet-DNP in 1990, pentacene has been the only and best option of triplet polarizing agents. However, the poor air-stability of pentacene has severely limited the applicability of triplet-DNP. We demonstrate the first example of air-stable polarizing agents with high polarizing ability comparable to pentacene (J. Phys. Chem. Lett., 2019, 10, 2208).
We also show the first example of triplet-DNP in water by downsizing the conventional bulk crystals to nanocrystals.

Speaker: Prof. Nobuhiro Yanai (Kyushu University)
• 11:55
DNP Polarizing Agents for High-Field, Fast-MAS and Variable Temperature 35m

MAS DNP is increasingly establishing itself as a powerful technique to boost sensitivity of NMR. It makes it possible to run, in minutes, experiments that would take weeks, or simply would not be possible otherwise because they are too insensitive. At 9.4 T (400 MHz) and 100 K, DNP enhancements of around 250 are now possible with several polarizing agents (PA) (AMUPol1, TEKPol2, and more recently(3) SPIROPOL,(4) PyPolPEG2OH,(5) bcTol,(6) AsymPolPOK,(7)…). Despite excellent performance at 9.4 T, these dinitroxide biradicals are much less efficient at 18.8 T, both in terms of enhancement and overall sensitivity gain. The need for more efficient PA stimulated the research of biradical with a narrow EPR line,(8) such as for trityl in TEMTriPOL.(9)
Here we show how either monoradical like BDPA, based on the Overhauser effect, or hybrid biradicals, based on the Cross-Effect mechanism and designed by coupling BDPA with a nitroxide unit, provide very high DNP enhancements and overall sensitivity gains at 18.8 T, with performance significantly exceeding dinitroxides at this field. Overhauser DNP with BDPA in OTP shows enhancements of over 100 at 18.8 T and 40 kHz MAS with 1.3 mm rotors at 100 K, with no depolarization or quenching effects.10 The enhancement also persists at higher temperature, with values of around 30 at –30 °C!(11)
Hybrid BDPA-nitroxide biradical show enhancements up to 185 at 18.8 T, 100K and 40 kHz MAS, which is so far the highest enhancement and sensitivity gain at high magnetic field.(12) We will discuss how the DNP performance of all these systems depends on a combination of several factors, from the magnetic properties of the polarizing agent to the role played by spin-diffusion. The potential of these polarizing agents for the characterization of pharmaceutical and functionalized material surfaces will be presented.

Speaker: Prof. Moreno Lelli (University of Florence)
• 12:30
Improving bis-nitroxides' geometry for MAS-DNP 30m

Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP) is becoming a mainstream method to increase the sensitivity of solid-state NMR experiments. By irradiating a sample with a strong microwave ($\mu$w) source, nitroxide biradicals are used to enhance the proton polarization via the cross-effect (CE) mechanism.$^1$
To increase the enhancement factor (the NMR signal ratio with/without $\mu$w irradiation, $\mathrm{\epsilon_{on/off}}$), the biradical structures underwent extensive optimization.$^{2, 3}$ In particular, the nitroxides’ g-tensors relative orientation ($\alpha,\beta,\gamma$) is essential for efficient CE under MAS.$^4$
From the early biradical design the role of $\beta$ was clearly identified experimentally and was recently confirmed theoretically by scanning the complete angular space.$^5$
Nonetheless both approaches (experimental and theoretical) are incomplete. First, improving $\mathrm{\epsilon_{on/off}}$ may not correlate with increasing proton polarization as biradical induces nuclear depolarization without $\mu$w. Biradicals with lower $\mathrm{\epsilon_{on/off}}$ can provide equal or better polarization performance.$^6$ Second, the simulations neglected the relative orientation’s impact on the enhancement vs magnetic field.$^7$ Therefore, the question “Can bis-nitroxide be improved?” is still debatable.
In this presentation, using a new quantitative theoretical approach,$^7$ this question is discussed and an ideal orientation is presented. A single parameter is sufficient to evaluate the potential performance of a bis-nitroxide structure. Finally, the method is illustrated on the bTurea series to explain recent experimental observation of AMUPol,$^2$ bcTol-M,$^3$ and HydrOPol.$^8$

1. Rosay et al., Phys. Chem. Chem. Phys. 12, (2010).
2. Sauvée et al., Chem. - A Eur. J. 22, (2016).
3. Geiger et al., Chem. - A Eur. J. 24, (2018).
4. Mentink-Vigier et al., J. Magn. Reson. 258, (2015).
5. Perras et al.,ChemPhysChem. 18, (2017).
6. Mentink-Vigier et al., J. Am. Chem. Soc. 140, (2018).
7. Mentink-Vigier et al., Phys. Chem. Chem. Phys. 21, (2019).
8. Stevanatoa et al., 60th ENC, Asilomar (2019).
Speaker: Frederic Mentink-Vigier (National High Magnetic Field Laboratory, Florida State University)
• 10:30 13:00
In-vivo: In-cell NMR: Session 29 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 10:30
In-Cell NMR: Past, Present and Future 35m

Recent breakthroughs in optical and electron microscopy have changed the fields of Cell and Structural Biology in a most profound manner, with ever more detailed information about the inner workings of cells becoming available. Complementary to the advancements, novel in situ methods are beginning to emerge as powerful tools in Cellular Structural Biology. Here, I discuss how recent developments in in-cell NMR and EPR contribute to our understanding of basic biological processes in live cells. Specifically, I outline how these techniques provide time-resolved atomic-resolution information about intracellular protein structures and functions, which cannot be obtained with any other method at this time.

Speaker: Prof. Phil Selenko (Weizmann Institute of Science)
• 11:05
In-cell PELDOR of spin-labelled RNA duplexes 25m

Structural investigation of nucleic acids is usually carried out either in diluted buffered solutions or on crystals of these biomolecules. These environments markedly differ from the native one in which these molecules are found, where effects as macromolecular crowding or intermolecular interactions can play a significant role on the conformation.

Aim of the present study was to investigate whether the structures of short RNA duplexes would be different when placed in a buffered solution vs inside Xenopus lævis oocytes. The structural information was obtained as a set of distance constraints between paramagnetic tags covalently linked to the biomolecule under study; these constraints were obtained from 4-pulse PELDOR measurements.

In this work two different labelling strategies were used to label a uridine with an isoindoline-derived spin label, differing both by the flexibility of the linker and by the point of attachment. In the first case, the spin label was post-synthetically conjugated to the ribose sugar ring via a thiourea linkage. In the second case, the spin label was attached to the uracil base through a C-C bond; an intramolecular hydrogen bond between the label and the uracil restricts the rotation around the C-C bond, resulting in a semi-rigid label. For both strategies, the paramagnetic centre was protected against the reducing environment of the cell by replacing the normal gem-dimethyl groups, flanking the nitroxide moiety, with gem-diethyl groups.

The results showed a clear, reproducible shift of the mean inter-spin distance to shorter values upon internalisation inside cells. In order to understand these findings, the duplexes were further investigated under different in-vitro conditions, such as in cytoplasmic extract from the Xenopus lævis oocytes as well as in the presence of protein crowders (BSA and lysozyme). The results of these experiments indicate an involvement of electrostatic interactions in the observed conformational changes.

Speaker: Dr Alberto Collauto (Institute of Physical and Theoretical Chemistry and Center for Biomolecular Resonance)
• 11:30
In-cell DNP Supported Solid-State NMR on Soluble Proteins 25m

Increasing evidence suggests that the highly complex and dynamic environment of the cell interior and its physiochemical setting imposes critical control on cellular functions, which is hardly reproducible under in vitro conditions. In-cell solution-state NMR can track such structural and dynamical interactions at the atomic level provided that proteins or other molecular units are small and tumble rapidly. On the other hand, solid-state NMR (ssNMR) has been used to probe proteins and large protein complexes in bacterial cells and at the cell membrane periphery of human cells. However, extending such studies to investigate proteins and molecular complexes inside human or bacterial cells poses additional challenges. Firstly, sample preparation schemes must be designed that achieve molecule-specific isotope labeling of biomolecules in cells at endogenously relevant protein concentrations. In addition, non-conventional NMR concepts must be used the overcome the inherent low sensitivity of such cell preparations. Dynamic Nuclear Polarization (DNP), has been widely used to boost sensitivity in ssNMR experiments. However, the strong reducing environment inside the cells can be deleterious to DNP radicals, thus precluding protein studies inside cells. We have developed tailored biochemical and solid-state NMR approaches that allow studying protein structure inside cells at atomic level under high-sensitivity DNP conditions. We demonstrate our methods on both Prokaryotic and Eukaryotic systems, thus opening up a plethora of applications for NMR-based cellular structural biochemistry.

Speaker: Mr Siddarth Narasimhan (Utrecht University)
• 11:55
Structure determination of antimicrobial peptides in model membranes and live bacteria 35m

Resistance to antibiotics is a growing health concern worldwide. Antimicrobial peptides (AMPs) present an alternative to conventional antibiotics but details of their mechanism of action and the basis for differences in their potency observed between different bacterial strains remain unclear. Structural information is crucial for defining the molecular mechanism by which these peptides recognize and interact with a particular lipid membrane. Nuclear magnetic resonance (NMR) structural investigations of cationic AMPs from Australian tree frogs in a range of different lipid systems will be discussed. Although these AMPs are unstructured in aqueous solution, they are alpha-helical in hydrophobic or membrane-like environments. The degree of helicity depends on membrane curvature and surface charge with a greater helical stretch in phospholipid bilayer membranes compared to micelles. Molecular dynamics simulations of the AMP, maculatin 1.1, show N-terminal exposure to the solvent and indicated that the peptide bends to adapt to the micelle curvature. Maculatin induced greater headgroup and acyl-chain perturbations for anionic phospholipids, which are found in bacterial membranes. The AMP appeared to lie on the surface of charged lipid membranes but insert in a transmembrane fashion with zwitterionic bilayer membranes. Solid-state NMR and dynamic nuclear polarization (DNP) studies of maculatin in live bacteria also support a transmembrane orientation. DNP NMR using spin-labelled peptides in combination with specifically 13C and 15N labelled maculatin give insight into pore formation and how AMPs self-assemble within bacterial membranes. The results of these structural studies could be used to design more potent AMPs for therapeutic applications.

Speaker: Prof. Frances Separovic (University of Melbourne)
• 12:30
Fluxomic studies by in cell and in vitro Dissolution-Dynamic Nuclear Polarization NMR 30m

Since the invention of dissolution Dynamic Nuclear Polarization (DDNP)$^{1}$ and the dramatic signal enhancement it provides in solution-state NMR, a wide range of applications have emerged. In particular, this improvement opens new avenues for the study of fast dynamical processes such as chemical reactions, with a time resolution smaller than a second. DDNP has proven of particular interest in the study of such metabolic processes as enzymatic reactions that are affected by many factors, such as dietary or environmental. These reactions can now be studied in vitro and in cell, providing access to the nature and degree of possible cell impairment.

We have initiated kinetic studies of enzymatic reactions using DDNP on the oxidative stage of the Pentose Phosphate Pathway (oxPPP)$^2$, one of the crucial metabolic pathways in cells. OxPPP produces NADPH, one of the main sources of reductive power in the cell. It comprises three enzymes: Glucose-6-Phosphate dehydrogenase (G6PDH), 6-Phosphogluconolactonase and 6-Phosphogluconic acid dehydrogenase.

By studying the enzymatic cascade involving Hexokinase and G6PDH, we could extract the relevant kinetic parameters involved in the process. The complexity of the kinetic pathway and the large number of model parameters makes it a particularly demanding task that requires the repeatability of the experiments. Fitting the signal build-up curves of the multiple metabolites allowed us to extract the relevant kinetic parameters. This showed satisfactory correlation with the enzyme activities used in the experiments.

In parallel, in cell experiments performed on E. coli allowed to provide a global ans quantitative view of Glucose metabolism in function of the cell-growth and cell stress conditions. In particular, the differential $\alpha / \beta$ anomeric Glucose uptake by the cells appeared as an indicator of the relative activities of Glycolysis and PPP.

1. Ardenkjær-Larsen et al. PNAS, (2003), 10158–10163.

2. A. Sadet et al. Chem.Eur.J. (2018), 4, 5456–5461.

Speaker: Mr David Guarin (Laboratoire des biomolécules, LBM, Département de chimie, École normale supérieure, PSL University, Sorbonne Université, CNRS, 75005 Paris, France)
• 10:30 13:00
Metabolomics: Session 27 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 10:30
New Discoveries on HBV infection by Metabolomics 35m

Hepatitis B Virus (HBV) is a double stranded DNA virus and belongs to hepadnavirdae family. HBV infection causes a severe liver infectious disease and has become a global problem affecting human health. At least 250 million people are chronically infected with HBV, with an estimated 650,000 deaths per year from HBV associated hepatocellular carcinoma, mainly in Asia. Current clinical strategy is aimed at inhibiting the HBV replication. Research in HBV infection is also hindered by the factor that there is no great animal models. We employed metabolomics approach and investigated both human circulating metabolites and cell model. We found that HBV replication induces the promotions of central carbon metabolism, biosynthesis of nucleotides and total fatty acids; HBV up-regulates the biosynthesis of hexosamine and phosphatidylcholine through activating glutamine-fructose-6-phosphate amidotransferase 1 (GFAT1) and choline kinase α (CHKA), respectively. Furthermore, we demonstrate that GFAT1 and CHKA are two potential targets for treating HBV infection. To elucidate the relationship between HBV replication and host downstream lipid metabolism, we measured 10 classes of phospholipids in HBV infected patients and cells and we found that the levels of phosphatidylcholine (PC), phosphatidylethanolamine, and lyso-phosphatidic acid were increased in HBsAg (+) group compared with HBsAg (-), while phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and sphingomyelin were decreased, which were confirmed in HBV infected HepG2.2.15 cell line. We further evaluated the levels of enzymes of PC pathways and found that PCYT1A and LPP1 for PC synthesis were up-regulated after HBV infection. Moreover, the HBV replication was inhibited when PCYT1A and LPP1 were knocked down. These results indicated that the PC synthesis in HBV infected host are regulated by PCYT1A and LPP1, which suggests that PCYT1A, LPP1 could be new potential targets for HBV treatment.

Speaker: Prof. Yulan Wang (Singapore Phenome Center, Lee Kong Chian School of Medicine, Nanyang Technological University)
• 11:05
Slice-selective, high-resolution MAS NMR of intact tissue biopsies gives improved spatial resolution of metabolic distributions 25m

Biological tissue biopsies are often heterogeneous in cell type and structure and the recognition of this heterogeneity is crucial for many diagnostic studies. For example, differentiation between involved and uninvolved tissue is paramount for the determination of exact tumour margins [DeFeo et al., 2010]. Conventional HR-MAS-NMR spectroscopy is an excellent tool to study metabolites and their abundances within such tissue biopsies [Beckonert et al., 2010; Lindon et al. 2009].
However, the resulting HR-MAS-NMR spectrum is an average over the entire biopsy and any information about underlying biological tissue heterogeneity lost.
We therefore aimed to implement a HR-MAS-NMR-based approach to explore an aspect of spatial heterogeneity of tissue biopsies.

We demonstrate that the previously established [Sarou-Kanian et al., 2015] combination of a gradient-assisted, slice-selective pulse sequence with the conventional HR-MAS-NMR setup and tissue sample preparation yields spatially resolved spectra within a single tissue biopsy of interest.
Slice-selective (SS) HR-MAS-NMR was established on a tissue biopsy of known spatial heterogeneity, namely chicken thigh muscle with skin, characterized by two distinct layers.

Despite short acquisition times of 2 min per selected slice, spectral resolution and sensitivity were excellent with a typical peak linewidth for alanine of ca. 1 Hz and an SNR of around 400 for a 28 scan experiment.

Further, we show that changes in intensity of individual metabolites can be tracked throughout the sample with respect to spatial origin. This allowed us to create a metabolite-specific, one-dimensional abundance profile throughout individual biopsies.
Resulting SS-HR-MAS-NMR spectra clearly show a distribution of metabolites within single tissue biopsies. Spectra from slices containing muscle clearly differed from those containing mostly skin, particularly so in lipid content.

Without modification to the standard hardware or sample preparation methods, SS-HR-MAS-NMR can be used to spatially resolve tissue biopsies with powerful implications for future studies far beyond cancer research.

Speaker: Ms Elisabeth V. Vonhof (Division of Integrative Systems and Digestive Medicine, Department of Surgery and Cancer, Imperial College London)
• 11:30
Advanced Analysis of Chronic Kidney Disease by NMR Derived Metabolomic Fingerprints 25m

Identification of chronic kidney disease patients at risk of progressing to end-stage renal disease (ESRD) is essential for treatment decision-making and clinical trial design. Here, we show that proton nuclear magnetic resonance (NMR) spectroscopy of blood plasma specimens together with techniques from machine learning improves the currently best performing kidney failure risk equation, the so-called Tangri score. Our NMR study cohort comprises 4640 participants from the German Chronic Kidney Disease (GCKD) study, of whom 185 (3.99%) progressed over a mean observation time of 3.70 ± 0.88 years to ESRD requiring either dialysis or transplantation. In this context, we also introduce mixed graphical models to reveal important associations between NMR derived metabolic features, demographic and drug features and variables measured by standard clinical chemistry. Results show important associations between chronic kidney disease and for example gout. In summary, we demonstrate that NMR substantially improves the analysis of chronic kidney disease.

Speaker: Prof. Wolfram Gronwald (Institute of Functional Genomics, University of Regensburg, Germany )
• 11:55
Improving survival predictability and biological insight through NMR based metabolomics of Acute Respiratory Distress Syndrome (ARDS) 35m

Acute Respiratory Distress Syndrome (ARDS), as characterized by the onset of clinically significant hypoxemia and diffuse pulmonary infiltrates, has been a challenge to the critical care physicians due to high death toll rate. Categorization of the severity of ARDS is based on degree of hypoxemia enumerated by partial pressure of oxygen to the fraction of inspired oxygen (PaO2/FIO2) ratio and chest X-ray. ARDS diagnostic criteria is based on Berlin definition and classified as mild ARDS (P/F between 200-300), moderate ARDS (P/F between 100-200), severe ARDS (P/F between <100). Due to complex etiology of ARDS, efforts are required to apply system biology tools to understand disease progression and to improve survival prediction. In this direction, we have applied nuclear magnetic resonance (NMR) based metabolomics to understand heterogeneous biology of ARDS. The NMR spectroscopy of mini – bronchoalveolar lavage fluid (mBALF) was optimised and several small molecular weight metabolites were identified which are indicator of lung pathology. NMR spectroscopy of human serum samples helps in identifying the metabolites associated with ARDS severity. Further sub classifying the progression, outcome and the metabolites contributing to pulmonary and non-pulmonary causes of ARDS, mBALF and serum samples were being used in larger sample size for which initial model was tested with respect to control showing good separation and accuracy. The sensitivity and specificity of individual serum metabolites and mBALF metabolites as resultant serum and mBALF endotypes were used further to determine their clinical predictability when combined with clinical APACHE and SOFA score. The accuracy increased to AUROC 1 indicating the clinical relevance of the above determined metabolic endotypes. Pathway analysis of serum endotype and mBALF endotype predictive of mortality gave important metabolic pathway symbolic of ARDS correlated changes in metabolism.

Speaker: Dr Neeraj Sinha (Centre of Biomedical Research)
• 12:30
The effect of osmolytes in biomolecular stability as investigated by NMR spectroscopy. Lessons from halophilic proteins 30m

Protein folding is usually driven by the hydrophobic core while the role of the surface residues is considered to be marginal. Intimately ligated to protein folding, protein stability establishes the energy required to unfold a protein and the equilibrium populations of the folded and unfolded conformations at a given temperature. Proteins from halophilic organisms challenge this concept since they are often unfolded at low salt conditions while they become folded only in the presence of high salt concentrations. This is possible thanks to a severe modulation of the amino acid composition in the surface of the protein. This halophilic signature is general and conserved and constitutes a biological meter for protein quinary structure (the protein stability and folding when considering the cosolute).
In here, we use NMR spectroscopy and other biophysical techniques to investigate the mechanisms contributing to protein haloadaptation (i. e. stabilization in KCl) and their potential interplay with the stabilization mechanism induced by osmolytes (Sarcosine, Taurine, TMAO, Sucrose, Glycine, Betaine and Trehalose), using several halophilic and mesophilic proteins as reporters. Our data suggest co-evolution of the surface residues to become stabilized not only by salt, but also by osmolytes as well.

Speaker: Dr Oscar Millet (CIC bioGUNE)
• 10:30 13:00
Solid-state NMR Methods: Session 30 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 10:30
High-Field and Fast-MAS Solid-State NMR: Enabling Application to Pharmaceuticals and Supramolecular Assembly 35m

Applications of advanced solid-state NMR methods for probing intermolecular interactions, notably hydrogen bonding are presented: Homonuclear 1H-1H double-quantum (DQ) experiments reveal proximities (typically under 3.5 Angstroms) among pairs of hydrogen atoms, for example distinguishing between ribbon-like or quartet-like self assembly in guanosine supramolecular structures [1-2] or pushing the limit of detection for a minority solid-state form of a pharmaceutical molecule [3]. 14N-1H spectra show one-bond NH connectivities or additionally longer-range NH proximities depending on the recoupling time employed: Applications to guanosine self assembly [4], a pharmaceutical [5] and to probe the stability of a fumarate salt [6] are shown.

1. Peters, G. M. et al J. Am. Chem. Soc. 2015, 137, 5819.
2. Reddy, G. N. M. et al Chem. Eur. J. 2017, 23, 2315.
3. Maruyoshi, K. et al J. Pharm. Sci. 2017, 106, 3372.
4. Reddy, G. N. M. et al Cryst. Growth Des. 2016, 15, 5945.
5. Tatton, A. S. et al Cryst. Growth Des. 2018, 18, 3339.
6. Corlett, E. K. et al CrystEngComm 2019, in press.
Speaker: Prof. Steven Brown (University of Warwick)
• 11:05
Protein resonance assignment without spectral analysis: five-dimensional spectroscopy of immobilized proteins at ultrafast MAS 25m

The difficulty to automate data acquisition and analysis of complex protein spectra has been one of the major bottlenecks for the widespread use of NMR spectroscopy in structural biology. A promising approach are spectra of high dimensionality (>3) which yield multiple nuclear correlations within fewer experiments, provide high resolution and unambigouos sequential resonance assignment, thus are prone to automation.
Multidimensional spectroscopy (5D-7D) has been explored in solution NMR, however, the concept suffers from a severe inherent contradiction: a satisfactory performance of multiple coherence-transfer experiments is only observed for globular proteins with molecular sizes smaller than about 20 kDa (fast tumbling) or by intrinsically disordered proteins. The deadlock is nowadays removed in proton-detected solid-state NMR at fast magic-angle spinning (MAS). Efficient multiple coherence transfers, narrow proton signals and high detection sensitivity, can be obtained, independently from molecular mass, employing high magnetic fields and ultrafast MAS. The application scope of high-dimensional spectroscopy is thus radically increased.
Here we employ Automated Projection SpectroscopY (APSY), which allows direct inference of a high-dimensional peak list from a number of lower order projection spectra (2D or 3D). We demonstrate the approach with two complementary 5D HN-detected experiments that evolve all traversed backbone nuclei: (H)NCOCANH and (H)NCACONH. We show that sensitive five-dimensional correlations are feasible on microcrystalline and fibrillar proteins at 60 and 110 kHz MAS. APSY, now embedded natively in Bruker TopSpin, not only handles data collection but also entirely bypasses spectral analysis. It delivers an output that directly contains the positions of all resonances. It is coupled to a flexible resonance assignment algorithm FLYA, yielding effortlessly expeditious resonance assignments. The protocol, automated from data collection up to resonance assignment, is in principle amenable to widespread access even by inexperienced spectroscopists, and may push forward the size limits of the proteins amenable to site-specific NMR studies.

Speaker: Dr Jan Stanek (Biological and Chemical Research Centre, Faculty of Chemistry, University of Warsaw)
• 11:30
Solid-state NMR strategies towards speed and resolution 25m

Solid-state NMR is a very flexible and powerful technique for the elucidation of geometry and dynamics information on a variety of samples. However, there is still a need to overcome sensitivity and resolution aspects along with the necessity to carry out multidimensional experiments in a short span of time. In order to overcome these challenges, we have made use of two approaches.

First approach involves the improvement of heteronuclear spin decoupling efficiency at high magic-angle spinning (MAS) frequencies. For this, a unified strategy of two-pulse based heteronuclear decoupling for high-spinning frequencies and low-power radio-frequency irradiation in solid-state MAS NMR is presented which incorporates simultaneous time and phase modulation. Decoupling sequences like TPPM, XiX and rCW turn out to be specific solution of this approach. This approach not only highlights the existing solutions but also generates new solutions for efficient decoupling.

Secondly, to speed up the data acquisition process, process, pulse sequences that implement sequential acquisition strategies on one and two radio radiofrequency channels with a combination of proton and carbon detection to record multiple experiments under MAS have been coded. These strategies are expected to work better with $^{1}$H detection under fast-magic angle spinning due to low RF amplitude requirements. $^{1}$H detection under fast MAS regime demands heteronuclear decoupling on $^{13}$C or $^{15}$N channel in order to achieve the maximum resolution. We show that the longitudinal $^{15}$N polarisation survives decoupling and can be used to perform multiple sequential experiments at fast MAS. Using multiplex phase cycling, we carried out numerous residue linking experiments in a single experimental block that is alone sufficient for obtaining assignments.

Speaker: Ms Kshama Sharma (TIFR Centre for Interdisciplinary Sciences)
• 11:55
Multiple acquisitions for protein assignment, structure and dynamics 35m

In this presentation I will discuss various implementations of multiple detection experiments for accelerating assignment, structure determination and relaxation measurements in the solid state NMR. Methods will include multiple receiver variants of time-shared TSAR experiments for obtaining long distance restraints, 1H-detected and 13C-detected experiments for spectral assignment compensated for the lower sensitivity of 13C detection and various implementations of sequential experiments for measuring simultaneously 15N and 13C' R1 and R. Various experimental consideration for multiple detection experiments will be discussed.

Speaker: Prof. Józef Lewandowski (University of Warwick)
• 12:30
Biomolecular structural conversion processes probed with DNP-enhanced, millisecond time-resolved solid state NMR 30m

We have developed experimental methods for initiating nonequilibrium structural conversion processes (e.g., protein folding, peptide self-assembly, ligand/receptor complex formation, etc.) by rapid mixing and for trapping intermediate states by rapid freezing after a defined time interval, on the millisecond time scale. When combined with low-temperature dynamic nuclear polarization, selective isotopic labeling, and solid state NMR techniques, these methods allow us to characterize the time-dependence of multiple aspects of molecular structure during a rapid structural conversion process. As an example, I will describe results for the folding and self-assembly of the 26-residue peptide melittin after a rapid pH jump. The data indicate that unstructured melittin monomers at low pH adopt helical conformations and self-assemble into antiparallel dimers at neutral pH in a cooperative manner on the 6-9 ms time scale. Melittin tetramers then form quickly, but become fully structurally ordered more slowly, on the time scale of about 60 ms. The latest results from other applications will also be described. Overall, this approach to studies of nonequilibrium structural conversions has broad applicability, providing information that is not readily available in as comprehensive a manner from alternative approaches such as optical spectroscopies.

Speaker: Dr Robert Tycko (National Institutes of Health)
• 13:00 14:00
Lunch 1h Harnack House

Harnack House

• 14:00 16:00
Posters: even numbered presentations Harnack House and Henry Ford Building

Harnack House and Henry Ford Building

• 15:50 16:10
Coffee 20m Harnack House

Harnack House

• 16:10 17:15
Biomolecules: Nucleic Acids: Session 33 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 16:10
Guanine-rich DNA regions and their amazing structures 35m

Double helix is the most known structure of DNA. It can account for transfer of genetic information. However, DNA can fold into a wide range of structures that are associated with its unique biological roles and functions. G-rich DNA segments adopting to d[G≥3N1–7G≥3N1–7G≥3N1–7G≥3] motif are populated in hundreds of thousands and have the potential to form a G-quadruplex structure. G-rich fragments from the PLEKHG3 gene can form tetrahelical structures that differ significantly from G-quadruplexes, despite containing the G-quadruplex folding motif d[G3NG3NG3NG3], where N=AGCGA. These sequences adopt tetrahelical cores of AGCGA repeats, connected with edge-type loops of G–G base pairs. A marked difference between G- and AGCGA-quadruplexes is their opposing response to changes in water activity. While the former become stabilized with decreasing water activity, the reverse is true for the latter (and B-DNA).
Another intriguing case when relying on sequence details alone to predict G-quadruplex structure was reported recently on a G-rich sequence found in the regulatory region of the RANKL gene, associated with homeostasis of bone metabolism. An oligonucleotide with four G-tracts of three successive guanine residues folds into a two-quartet basket-type G-quadruplex.
d[(G4C2)3G4] implicated in neurological disorders ALS and FTD forms two major G-quadruplex structures. Structural characterization of the G-quadruplex named AQU revealed an antiparallel fold composed of four G-quartets and three lateral C–C loops. Two C•C base pairs are stacked on the nearby G-quartet and are involved in a dynamic equilibrium between symmetric N3-amino and carbonyl-amino geometries and protonated C+•C state.

Selected references: Angew. Chem. Int. Ed. 2019, 58, 2387. Angew. Chem. Int. Ed. 2018, 57, 15395. Nucleic Acids Res. 2018, 46, 11605. Nucleic Acids Res. 2019, 47, 2641. J. Am. Chem. Soc. 2019, 141, 2594. Nucleic Acids Res. 2018, 46, 4301. J. Am. Chem. Soc. 2018, 140, 5774.

Speaker: Prof. Janez Plavec (Slovenian NMR Center, National Institute of Chemistry, Ljubljana, Slovenia, Slovenia)
• 16:45
Unique quadruplex structure and anti-disease activity of RNA aptamer, and in-cell NMR of nucleic acids 25m

We previously identified an RNA aptamer against a prion protein, r(GGAGGAGGAGGA) (R12). We showed that R12 forms a unique quadruplex structure and reduces a level of the abnormal prion protein, PrPSc, in the mouse neuronal cells, implying its therapeutic potential as to prion diseases (1,2). We also utilized R12 to develop the K+-responsive ribozyme (3) and RNA aptamer against HIV-1 Tat protein (4), using its quadruplex formation in response to K+. Here, we reveal that RNA with analogous sequence to R12 can reduce the level of PrPSc much more efficiently. Structure determination rationalizes the higher anti-prion activity of this new RNA (5).

Some recent studies suggested that amyloid beta (Aβ) forms soluble oligomers, protofibrils and fibrils; the Aβ oligomers being more toxic than the fibrils. The Aβ oligomers reportedly bind to prion protein (PrP), which acts as a receptor on the cell membrane, possibly resulting in Alzheimer’s disease (AD) (6). Thus, it is thought that compounds that can disrupt the formation of the prion-Aβ oligomer complex may prevent AD. Here, we demonstrate that R12 inhibits the interaction of PrP with Aβ, which implies therapeutic potential of R12 to AD (7).

In-cell NMR is a promising method to obtain the information on the structure, dynamics and interaction of biomolecules. We succeeded in observing NMR signals of DNA/RNA in living human cells for the first time (8). The observed signals directly suggested the formation of DNA/RNA hairpin structures in living human cells. Further development of in-cell NMR studies of nucleic acids in human cells will be presented.

1) Nucleic Acids Res., 2013. 2) Nucleic Acids Res., 2014. 3) Chem. Commun., 2015.
4) Chem. Commun., 2017. 5) submitted. 6) Nature, 2009. 7) FEBS J., 2019. 8) PCCP, 2018.

Speaker: Prof. Masato Katahira (Kyoto University)
• 16:10 17:15
EPR: Session 31 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 16:10
Muti-Extreme THz ESR -Recent Developments and Future- 35m

THz ESR under multi-extreme conditions, which covers the frequency region between 0.03 and 7 THz1, the temperature region between 1.8 and 300 K1, the magnetic field region up to 55 T1, and the pressure up to 1.5 GPa2, has been developed in Kobe. Firstly, we will show our recent developments of the torque magnetometry3 and mechanically detected ESR4 measurements using a commercially available membrane-type surface stress sensor, which is the extension from our micro-cantilever ESR5, and its application to the microliter solution sample (myoglobin)6. Secondly, we will show that the pressure region is extended to 2.7 GPa using the hybrid-type pressure cell7. Recent development of high-pressure THz ESR with the 25 T superconducting magnet8 and its application to Cs2CuCl4 will be discussed9.
1. H. Ohta et al., J. Low Temp. Phys. 2013, 170, 511.
2. T. Sakurai et al., Rev. Sci. Inst. 2007, 78, 065107
3. H. Takahashi et al., J. Phys. Soc. Jpn. 2017, 86, 063002 (Editor's Choice)
4. H. Takahashi et al., Rev. Sci. Intrum. 2018, 89, 036108
5. H. Ohta et al., AIP Conf. Proceedings 2006, 850, 1643; E. Ohmichi et al., Rev. Sci. Instrum. 2008, 79, 103903; E. Ohmichi et al., Rev. Sci. Instrum. 2009, 80, 013904; E. Ohmichi et al., J. Mag. Res. 2013, 227, 9; H. Takahashi, E. Ohmichi, H. Ohta, Appl. Phys. Lett. 2015, 107, 182405.
6. T. Okamoto et al., Appl. Phys. Lett. 2018, 113 223702 (Editors Picks)
7. K. Fujimoto et al., Appl. Mag. Res. 2013, 44, 893; H. Ohta et al., J. Phys. Chem. B 2015, 119, 13755; T. Sakurai et al., J. Mag. Res., 2015, 259,108; T. Sakurai et al., J. Phys. Soc. Jpn. 2018, 87, 033701.
8. T. Sakurai et al., J. Mag. Res. 2018, 296, 1-4
9. S. A. Zvyagin et al., Nature Communications, 2019, 10,1064

Speaker: Prof. Hitoshi Ohta (Kobe University, Molecular Photoscience Research Center)
• 16:45
High-Q Photonic Band Gap Resonators for mm-wave EPR of Lossy Aqueous Samples and Thin Films 25m

Water and other polar molecules are known to absorb electromagnetic radiation and the absorption is particularly strong in the mm-Wave (mmW) range. Metal surfaces are also becoming increasingly lossy. These high dielectric losses represent the major challenge for constructing EPR and also DNP NMR probeheads suitable for accommodating samples with the maximum volume. While large samples can be fitted into non-resonant mmW structures, resonator cavities offer significantly higher mm-wave B1 fields – an essential condition for DNP. High-Q resonators also provide the best EPR concentration sensitivity at X- and Q-band. Last year we described a radically new line of EPR resonators that are based on one-dimensional photonic band gap (PBG) dielectric crystals. PBG crystals were assembled from λ/4 low-loss dielectric layers with alternating dielectric constants and demonstrated experimental Q≈520 at 94.3 GHz. Anodic aluminum oxide nanoporous disc of 50 μm in thickness was employed as an aqueous sample holder allowing for ca. 2-3 μl sample volume. Here we report on significant improvements of PBG EPR resonators in both sample volume and experimental Q-factors while minimizing dielectric losses even for liquid aqueous samples. A series of smooth and corrugated 95 GHz transitions were tested to improve flatness of the mmW front. The resonator Q-factor was further improved by increasing the sample diameter from 12 to 36 mm yielding 9-fold sample volume increase. The best experimental Q≈3,300 has been observed for 8 alternating λ/4 layers of alumina and air. Experimental tests of the new resonators for aqueous and thin film samples are also reported. Finesse of 200 GHz PBG resonator for 300 MHz (1H) DNP was improved by forming photonic crystals from dielectric layers with high ε12 ratio. Supported by NIH R21EB024110 and R01GM130821.

Speaker: Prof. Alex Smirnov (North Carolina State University)
• 16:10 17:15
In-vivo: Session 35 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 16:10
Imaging Human Brain Function and Connectivity over Multiple Spatial Scales 35m

An extraordinary feature of brain function is the encoding of information at multiple spatial and temporal scales, going from the cellular level in the form of action potentials to coordinated activity over billions of neurons spanning large parts of the brain, if not the entire brain, to achieve perception and behavior. Bridging and spanning these multiple scales of organization is an essential, but a daunting task necessary for understanding brain function and ultimately dysfunction. Rapid developments in instrumentation for RF transmission and signal detection, a push to exploit unique advantages available at very high magnetic fields (achieving 7 Tesla in 1999 and currently at 10.5T for human imaging), despite the major challenges of imaging at the correspondingly high RF frequencies, and a plethora of novel imaging acquisition techniques that increase spatiotemporal sampling has been bringing transformative changes into our ability to map human brain function and connectivity. These developments complemented by other non-MR imaging methods hold the promise that in the near future it will be feasible to integrate information from the level of a single synapse to whole brain networks that define behavior.

Speaker: Prof. Kamil Ugurbil (University of Minnesota)
• 16:45
Deuterium Metabolic Imaging for in-vivo monitoring of pregnancy in mice at 15.2 T. 25m

The roster of molecular imaging methods has been recently extended by the introduction of deuterium metabolic imaging (DMI), whereby after administration of a deuterated precursor in rodents or humans, deuterium MRS and MRSI is used to examine metabolic products such as glutamine/glutamate or lactate when applied to brain studies. Here we examine its use for imaging pregnancy-related conditions such as preeclampsia and intrauterine growth restriction. Late-term pregnant ICR mice at around day 19 of gestation were administered with either uniformly deuterated glucose or glucose selectively doubly deuterated on C6 position intravenously at a dose of 2.3 g/kg body-weight. Deuterium chemical-shift imaging (CSI) and non-localized MRS were then performed on a Bruker-Biospec at 15.2T with a “sandwich setup” whereby a 20x45 mm Bruker 1H butterfly surface coil was placed underneath the mouse abdomen, and a customized 20 mm single-loop surface coil tuned to deuterium at 99.8 MHz placed on top of the mouse belly. Slice-selective 1H anatomical and 2H CSI data were then acquired; the latter delivered one 3D data set every 7 min with a FA 90 deg, TR 100 ms, slice thickness 7 mm, FOV 45x45 mm, and a matrix size of 8x8, which was interpolated to 32x32 elements.
Deuterated glucose was predominantly observed localized in the maternal kidney, dropping significantly after 40 min while having its peak in the entire fetal tissue. As a main product of metabolism HDO was observed, formed mostly in the fetuses and with an intensity that grew even 3h after injection. Although of low S/N, lactate maps indicate maximum lactate production 50-70 min after injection localized in fetal livers.
We conclude that DMI may offer new ways of prolonged molecular imaging for monitoring of pregnancy conditions at thermal polarization. Further animal studies of these models as well as of cancer models are ongoing.

Speaker: Dr Stefan Markovic (Weizmann Institute of Science, Department of Chemical and Biological Physics, Rehovot, Israel)
• 16:10 17:15
Metabolomics: Methods: Session 34 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 16:10
A Tale of Two Sugars: 13C NMR Tracking of the Metabolic Fates of Glucose and Fructose in Cancer 35m

Increased dietary consumption of sugar has been implicated in a number of clinical pathologies, including obesity and other metabolic diseases. High fructose corn syrup, a sugar mixture of about 40% glucose and 60% fructose, is a ubiquitous sweetening additive in a number of drinks and food. In this study, we have investigated the metabolism of these two types of sugar in SfXL glioblastoma and HuH7 hepatocarcinoma cell lines. 13C NMR spectroscopy was used in this study due to high specificity courtesy of the wide chemical shift dispersion of carbon-13. The goal of this study was to investigate the metabolism of fructose and glucose in brain and liver cancer, given the ubiquity of these two sugars in Western diet and the high sugar addiction of these cancers. The main finding of this preliminary work is that, despite the same caloric content of these two sugars, fructose and glucose metabolized quite differently in brain and liver cancer cells. In the absence of glucose in the media, there was no indication of metabolism of [U-13C6]fructose in SfXL cells. In the presence of unlabeled glucose in DMEM, we have observed metabolism of [1-13C]fructose into [3-13C]lactate. However, lactic acid production rate from [1-13C]fructose is found to relatively slower compared to lactic acid production from [U-13C6]glucose. On the other hand, substantial lactic acid production from [U-13C6]fructose was observed in HuH7 liver cancer cells due to the presence of specialized hepatic enzymes that can metabolize fructose. Metabolic kinetics of these two sugars as well as the NMR results of co-administered 13C-fructose and 13C-glucose will be presented.

Speaker: Prof. Lloyd Lumata (University of Texas at Dallas)
• 16:45
Metabolic Pathway Profiling (MPP) with stable-isotope tracing 25m

Stable isotope tracers such as $^{13}$C are increasingly being used to study metabolism in high resolution. However, determining the metabolic fluxes within the system remains technically challenging due to both the complexity of the metabolic network and the sophisticated methods required to analyse the complex spectra derived from NMR and mass spectrometry. Here we present three tools to aid and expand the analysis of tracer based metabolism studies.

Firstly we have recently developed an algorithm (CANMS) that allows the simultaneous analysis of NMR and MS data, which yields a model-free isotopomer distribution, thus allowing a better understanding of metabolic mechanisms. We also demonstrate improved NMR experiments that allow more rapid data acquisition of 2D-$^1$H,$^{13}$C-HSQC NMR spectra and the determination of per carbon $^{13}$C percentage incorporation via $^{12}$C filtered 1D-$^1$H-NMR spectra. These experimental approaches allow us to accurately determine the amount of $^{13}$C incorporated into metabolites from a single sample. This is vital in order to be able to reliably use this methodology in a clinical setting or in vivo.

Secondly, the manual analysis of 2D-NMR spectra is a laborious process which can lead to biased and inconsistent results. To retrieve meaningful information, we have developed a data-driven algorithm to annotate and analyse multiplets in 2D-$^1$H,$^{13}$C-HSQC NMR spectra arising from $^{13}$C-$^{13}$C scalar couplings. The algorithm performs accurate metabolite pick-peaking and multiplet analysis, determining the contribution of each multiplet component to the metabolite signal.

Lastly, we present improved pulse sequences that allow the detection of certain quaternary carbon nuclei thus increasing the information content available and therefore reducing the probability of over-fitting the data. Using our innovative approach, we are able to correlate tracer data with metabolic pathway activity in our cell line model, which is then compared with the clinical outcome of tracing perfused, pre-transplant kidneys.

Speaker: Dr Christian Ludwig (University of Birmingham)
• 16:10 17:15
Spin Physics: Session 32 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 16:10
Up-conversion of NMR signals from radio-frequency to optical regimes through a mechanical transducer 35m

Conventional reception of NMR signals relies on electrical amplification of the electromotive force caused by nuclear induction. In general, the signals cannot be transported without noise being added through the process of amplification before being acquired.

Here, we report a different approach that potentially leads to much less noise added through signal transduction. The idea is to employ up-conversion of radio-frequency NMR signals to an optical regime using a high-stress silicon nitride membrane that interfaces the NMR-probe circuit and an optical cavity. In this approach that we call Electro-Mechano-Optical NMR, or EMO NMR [1-2], A metal layer coated on the membrane serves both as an electrode of a capacitor and a mirror of an optical cavity.

• the nuclear induction signal is transcribed to the vibration of the membrane through the electro-mechanical coupling.
• In turn, the displacement of the membrane modulates the light in the optical cavity due to the opto-mechanical coupling.

In this way, optical NMR detection is realized without sacrificing the versatility of the traditional nuclear induction approach. Theories predicts that the added noise through the EMO scheme can be made smaller compared to that in the conventional NMR.

In the presentation we show demonstration of EMO NMR as well as our current efforts toward its extension, including:

• Rf-to-light signal up-conversion using a Hihg-temperature superconducting rf resonator,
• New design and fabrication of a compact rf-to-light transducer that would fit inside the bore of a superconducting magnet, and
• Development toward combination to EMO MRI and MAS.

[1] K. Takeda, K. Nagasaka, A. Noguchi, R. Yamazaki, Y. Nakamura, E. Iwase, J.M. Taylor, K. Usami, Optica. 5 (2018) 152. doi:10.1364/OPTICA.5.000152.
[2] Y. Tominaga, K. Nagasaka, K. Usami, K. Takeda, J. Magn. Reson. 298 (2019) 6–15. doi:10.1016/j.jmr.2018.11.003.

Speaker: Dr Kazuyuki Takeda (Kyoto University)
• 16:45
Algorithmic cooling by using long-lived singlet states 25m

Algorithmic cooling is a relatively new method to increase overall spin-polarization in NMR, which is based on manipulations of coupled slow-relaxing and fast-relaxing spins. The method enables increasing the magnetization of slow-relaxing spin by using the ability of fast-relaxing spins to pump entropy into the environment. Here, we suggest a new method to increase spin polarization by using long-lived spin order. In the simplest case of a two-spin system, such a spin order is singlet order, while the fast-relaxing order is represented by spin magnetization.
We have developed a novel approach to algorithmic cooling by using a strongly-coupled spin pair, in which the long-lived singlet order can be sustained even in the absence of spin-locking. Experiments are performed for a naphthalene derivative having a pair of 13C labels with extremely long-lived singlet order. Algorithmic cooling is achieved by performing selective singlet-to-T+ and singlet-to-T conversion steps multiple times and allowing magnetization relaxes between subsequent conversions.
Firstly, we achieved the efficiency of singlet order formation of 0.82 (measured in units of thermal magnetization), which is greater than 2/3, which is the maximal theoretically allowed value assuming spin evolution given by unitary transformations. Secondly, we managed to increase the magnetization of the two-spin system by a factor of 1.23. For achieving such a conversion efficiency, we optimized methods used for singlet order generation, M2S, APSOC, and adiabatic SLIC.
To describe the new phenomena, we implemented a theoretical approach. In the simplest variant, we assume that lifetime of singlet order much longer than the T1-relaxation time and calculate the maximal theoretically allowed efficiency of algorithmic cooling. A more advanced superoperator-based approach enables quantitative modeling of the experiments: the theoretical result is in a good agreement with experimental findings.
The reported study was funded by RFBR according to the research project №19-32-80004

Speaker: Bogdan Rodin (International Tomography Center)
• 17:15 17:55
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 17:15
Pulsed magnetic resonance with a free-electron laser 40m

In the highest-field NMR magnets (currently 23.5 T, 1 GHz proton NMR frequency), the Larmor precession frequency for spin-1/2 electrons is 660 GHz. The recently-demonstrated 32 T superconducting magnet at MagLab in Tallahassee pushes the Larmor frequency to nearly 900 GHz. However, at the present time, it is difficult to generate a programmable sequence of phase-coherent narrow-band pulses with kW peak powers above 100 GHz (3.5 T), precluding the rapid coherent manipulation of electron spins that is required for high-power pulsed EPR, pulsed electron-nuclear double resonance (ENDOR), and pulsed dynamic nuclear polarization (DNP)-enhanced NMR in modern NMR spectrometers. The UC Santa Barbara Free-Electron Lasers (FELs), which generate high-power quasi-continuous-wave (cw) pulses between 0.24 and 4.5 THz, are now being used to drive a pulsed EPR spectrometer at 8.6 T (240 GHz). This talk will include a discussion of methods we have developed for converting the FEL output into a sequence of one or two pulses with durations as short as a few ns, resonator-free π/2 times below 10 ns, and, recently, multi-step phase-cycling. These pulse sequences, together with a home-built EPR spectrometer, have enabled measurements including Rabi oscillations, longitudinal and transverse relaxation times, and “instantaneous spectral diffusion” in systems including Nitrogen impurities (P1 centers) in diamond, and stable free radicals in both solid and solution phases. Current efforts to implement FEL-powered pulsed DNP and to generate more complex pulse sequences at 240 GHz will be described. Finally, I will discuss the outlook and scientific opportunities for FEL-powered EPR, DNP, and ENDOR at fields up to 30.5 T (1.3 GHz proton-NMR frequency, 854 GHz electron Larmor frequency). This work is supported by the NSF under grants DMR-1626681 and MCB-1617025.

Speaker: Prof. Mark Sherwin (University of California at Santa Barbara)
• 19:00 21:30
Conference Banquet 2h 30m
• Friday, 30 August
• 08:40 10:00
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 08:40
NMR of Aromatic Side Chains in Large Proteins 40m

Current NMR methods for studying proteins are primarily focused on backbone resonances and on methyl bearing side chains. In contrast, NMR of aromatic side chains has been less pursued although these moieties form a large portion of the hydrophobic protein cores. This is in part due to the complexity of aromatic side-chain spectra, which appear in a narrow and crowded spectral region. 13C-dispersion of spectra reduces the complexity but the large carbon-carbon and carbon-proton couplings make spectra difficult to analyze. The TROSY effect of aromatic carbons has been realized early on as an approach to make aromatic spin systems more accessible, and alternate 13C labeling with judiciously chosen pyruvate precursors can render rather well resolved aromatic spectra that can yield NOE contacts with and between aromatic side chains in larger proteins. With the ability to introduce 19F-labeled aromatic residues into proteins, we explored whether we could utilize the large CSA of fluorine to create a 19F-13C TROSY effect for more efficient detection of aromatic signals in large proteins. Indeed, simulation of the relaxation properties using the Spinach program indicated that 13C detected FC TROSY experiments can yield very sharp signals but need specific labeling strategies. Similar advances can also be made with 19F-13C labeled nucleotide bases, some of which promise to yield very sharp TROSY signals that are expected to deteriorate little with larger systems. NMR access to mobility and interactions of aromatic side chains is particularly interesting for elucidating mechanisms of signal transduction of membrane protein receptors where binding of agonists or antagonists may cause small structural and dynamic changes to define signaling.

Speaker: Prof. Gerhard Wagner (Harvard Medical School)
• 09:20
Manipulation of Spin Dynamics for Extraction of Spectral Parameters, Ultra High Resolution and Sensitivity Enhancement: Application to small molecules 40m

The smaller chemical shift dispersion and the pairwise interaction among all the abundant nuclear spins of the molecule renders 1H NMR spectrum highly complex, severely hindering the straightforward analysis and the accurate determination of homo- and hetero- nuclear scalar couplings. The inherent insentitivy of NMR technique poses additional challenge. We have manipulated the spin dynamics and designed number of two dimensional techniques to partially address these problems. The developed NMR methodologies lead to the extraction of spectral parameters, resulted in ultra high resolution and enhanced sensitivity of 1H detection. My talk will be focused on the application of some of our experimental methods to derive specific information on the small molecules. The special emphasis will be on our novel techniqques, QG-SERF, Clean-G-SERF and PS-Clean-G-SERF. The QG-SERF permitted the rapid determiantion of spectral information, while the other two techniques completely eradicates the axial peaks and suppress the evolution of unwanted couplings while retaining only the couplings to the selectively excited proton thereby facilitating the accurate determination of indirect spin-spin couplings even from a complex proton NMR spectrum in an orchestrated manner.

Speaker: Prof. Nagaraja rao Suryaprakash (Indian Institute of Science)
• 10:00 10:30
Coffee 30m
• 10:30 12:30
Dynamics: Session 38 Lecture Hall D

Lecture Hall D

Henry Ford Building

• 10:30
Allosteric signaling pathways and energetics in the glucocorticoid receptor 35m

The glucocorticoid receptor (GR) binds steroid hormones, leading to structural rearrangements that drive DNA binding, recruitment of coregulator proteins, and ultimately gene regulation. Different receptor-ligand complexes have distinct interactions with coregulators, resulting in differential gene regulation. The allosteric mechanism within the ligand-binding domain (LBD) has remained unknown. We used a combination of NMR relaxation dispersion and surface plasmon resonance to delineate allosteric signaling pathways within the LBD and their energetics. CPMG dispersions map out a dynamic network of residues linking the ligand-binding pocket to the activation function-2 interface, where helix 12, a switch for transcriptional activation, undergoes ligand and coregulator dependent conformational exchange dynamics. We quantified the observed variation in coregulator affinity and helix 12 activation in terms of free energies of allostery between sites. The results pinpoint how differential activation of GR arises from gradual population shifts within a dynamic ensemble of conformations.

Speaker: Prof. Mikael Akke (Lund University)
• 11:05
Rheological NMR for the study of polymer dynamics 25m

Rheo NMR has been applied to investigate the effect of external shear on the aggregation and on the chain dynamics of polymers. A Couette cell with the polymer melt or solution in the gap is applied. To get further insight, oscillating rotation in addition to continuous rotation has been applied.
An in-house built rheo NMR system using a servo motor, avoiding any vibrations has been used on a spectrometer with a microimaging accessory. For the oscillatory shear a crank mechanism has been introduced between the motor and the drive of the Couette rotor.
The combination of PFG NMR with imaging enables measuring the flow profiles In particular for low-viscosity liquids like dilute solutions or lower molecular weight polymers flow pattern deviating from the expected linear velocity gradient are observed. Around the turning point in the oscillatory shear counterflow is observed which apparently leads temporarily to a shear rate larger than the gap averaged shear rate applied when the angular velocity is at its peak value. This effect strongly depends on the viscosity.
In particular the spin-spin relaxation time T2 measured via CPMG is sensitive to the polymer chain segment motion. The expected shear-induced order in polymer melts and solutions is detected for very low molecular weight (oligomers) only. For higher molecular weight the loss or rearrangement of entanglements leading to longer chain segments between the entanglements and thus enhanced polymer dynamics manifested in longer T2 is the dominating effect.
This has been observed for both continuous and oscillatory shear at large shear angles. Varying the angle in the oscillatory shear in addition enables to study the onset of this change of the polymer chain motion and thus the onset of the rearrangement of the entangled polymers.

Speaker: Dr Ulrich Scheler (Leibniz-Institut für Polymerforschung Dresdne e.V.)
• 11:30
Molecular ordering and dynamics in anisotropic soft materials studied by low-resolution proton NMR 25m

With the advent of site-specific isotope labeling and deuteration, the study of local dynamics in biological macromolecules, has reached levels of unprecedented accuracy. There are, however, many cases in e.g. soft materials science, where such strategies are not feasible. While classical carbon-13-based solid-state NMR techniques are often possible, they nevertheless suffer from low sensitivity at natural abundance. Often, proton low-resolution time-domain NMR is fully sufficient, in particular when dealing with materials consisting of identical repeat units. Specifically, multiple-quantum NMR has been established as the most quantitative approach to extract order parameters and correlation times and assess the dynamic heterogeneity in such systems [1]. This is despite of data analyses being restricted to rather simple theories approximating the multi-spin dipolar coupling situation in terms of a second moment. While the methodology can be considered mature for the case of isotropic samples, challenges arise for oriented samples. Here, we highlight a recent methodological advance concerning the order parameter analysis of oriented materials with uniaxial dynamics, tested on liquid crystals [2] and now applied to the investigation of local chain stretching in mechanically deformed swollen elastomers. The latter project extends our earlier work concerned with the molecular-level deformation of polymer chains in bulk elastomers [3].

[1] K. Saalwächter, in: G.A. Webb (ed.), Modern Magnetic Resonance,DOI 10.1007/978-3-319-28275-6_59-2, Springer 2017
[2] A. Naumova, C. Tschierske, K. Saalwächter, Solid State Nucl. Magn. Reson. 2017, 82-83, 22
[3] M. Ott et al., Macromolecules 2014, 47, 7597

Speaker: Prof. Kay Saalwächter (Martin-Luther-Univ. Halle-Wittenberg)
• 11:55
Extending the range of magnetic fields for high-resolution biomolecular NMR by orders of magnitude 35m

Higher magnetic fields lead to higher sensitivity and higher resolution. Reaching higher fields is key to study biomolecular systems of increasing complexity. Yet, higher fields are not optimal for all applications of NMR, neither for all nuclei. For instance, the chemical shift anisotropies of carbon-13 nuclei in many chemical entities, or that of fluorine-19 lead to transverse relaxation rates incompatible with efficient NMR experiments of large biomolecules and assemblies at the highest magnetic fields. On the other hand, low magnetic fields provide rich information on molecular dynamics, as demonstrated by relaxometry, but are generally associated with sensitivity and resolution incompatible with site-specific studies of biomolecules. A dilemma for biomolecular NMR is: how can we benefit from the highest magnetic fields available while optimizing the field-dependent sensitivity, resolution and information of most NMR experiments?

The solution to this dilemma is to couple high-field NMR with low- or variable-field NMR in a single spectrometer. We use a sample shuttle to displace the NMR sample in the stray field of a high-field magnet to explore low magnetic fields in the course of an NMR experiment, while keeping polarization and detection at high field for sensitivity and resolution. In addition, the sample shuttle couples a magnetic center at 0.33 T with a magnetic center at 14.1 T in a two-field NMR spectrometer. This system allows us to perform pulse sequences, where each part is performed at the most optimal of the two fields. We will show a series of applications to the determination of site-specific protein dynamics on nanosecond timescales as well as a series of examples of two-field NMR experiments that provide more efficiency or information than the equivalent high-field-only experiment. Two-field NMR spectroscopy opens a route to boost the potential of high-resolution biomolecular NMR.

Speaker: Prof. Fabien Ferrage (CNRS and Ecole Normale Superieure)
• 10:30 12:30
Hyperpolarization: Session 39 Lecture Hall A

Lecture Hall A

Henry Ford Building

• 10:30
High Throughput Hyperpolarization for Drug Screening 35m

Many limitations of state-of-the-art drug screening by nuclear magnetic resonance (NMR) can be overcome by means of high-throughput hyperpolarization. There is an urgent need for innovative experimental screening techniques to identify new drugs as the resistance of « superbugs » against known drugs, e.g., against mycobacterium tuberculosis and other pathogens. Screening techniques must be capable of ranking promising drug candidates (“ligands”) according to their affinity for a protein, a nucleic acid, or a macromolecular complex (“targets”), in order to inhibit their function. Ligand-based NMR methods can monitor parameters such as chemical shifts, diffusion coefficients, dissociation constants KD, and kinetic kon and koff rates. NMR is particularly powerful to identify weakly binding ligands, which are crucial for fragment-based drug discovery (FBDD). However, even when boosted by current Dynamic Nuclear Polarization (DNP) methods, NMR is exceedingly slow and cumbersome. Our team is working towards the transformation of DNP-enhanced NMR into a competitive method for drug screening by introducing several ground-breaking innovations: multiplexed hyperpolarisation, high-speed transfer of frozen droplets, in situ dissolution, multiplexed detection using a stack of microfluidic detection chambers, and improved contrast due to long-lived states (LLS) of nuclei such as 19F and, more surprisingly, 2H in deuterated heavy drugs.

Speaker: Prof. Geoffrey Bodenhausen (Ecole Normale Supérieure)
• 11:05
Frequency-Chirped Millimeter-Wave Control of 13C-DNP in Diamond 25m

Electron and nuclear spins in diamond have long coherence and relaxation times at room temperature, making them a promising platform for applications such as biomedical and molecular imaging and nanoscale magnetic field sensing. While the optically-active nitrogen-vacancy (NV) defect has received a great deal of attention, the substitutional nitrogen (or P1) center also exhibits long coherence and relaxation times. These P1 centers are typically present at significantly larger concentrations (about an order magnitude larger) than NVs, allowing us to explore the role of P1-P1 interactions in mediating DNP. The system can, in principle, show DNP via the solid effect (SE), cross effect (CE) and Overhauser effect (OE) depending on the P1 concentration and the field.

Here, we show enhancement of natural abundance 13C nuclei found within the diamond, using the unpaired electron of the P1 center (concentration 110-130 ppm) in particles with a 15-25 μm diameter, under static conditions at room temperature and 3.4 T. From the DNP spectrum we conclude that both the SE-DNP and OE-DNP mechanisms are active. The OE, in our case, results in negative enhancement, in contrast to previous results reporting positive OE enhancements. A negative OE implies that zero-quantum relaxation is more effective than double-quantum relaxation, likely due to strong anisotropic hyperfine interactions. We also explore the effect of frequency modulation (FM) of the DNP mechanism. Preliminary results suggest that the OE benefits from faster FM (>100 kHz) whereas the SE does not. This suggests that we can control which DNP mechanism is effective using FM parameters such as frequency, amplitude and shape.

Speaker: Dr Daphna Shimon (Dartmouth College)
• 11:30
Signal-improved real-time NMR spectroscopy of proteins by hyperpolarized water 25m

Hyperpolarized water produced by dissolution dynamic nuclear polarization (dDNP) has recently been shown to enable the detection of hyperpolarized spectra of proteins with up to 300-fold improvement in signal amplitudes. With this dDNP approach, novel insights can be gained into solvent accessible surfaces, ligand interactions, and complex protein geometries. Examples of applications to host-ligand systems including peptides and folded as well as intrinsically disordered proteins (IDPs) have demonstrated the broad applicability of the hyperpolarized water approach. [1-5]

In this contribution, we present recent efforts to combine dDNP with real-time NMR, aimed at tracking protein-ligand binding events and protein-solvent interactions at a sub-Hertz sampling rate. Two applications will be presented: (1) The use of hyperpolarized water to examine the kinetics underlying protein-ligand interactions. Here, non-equilibrium dynamics in the osteopontin-heparin host-ligand system were monitored in a dDNP experiment by simultaneous mixing of the protein with the ligand and hyperpolarized water. (2) A proof-of-concept for real-time protein dDNP at residue-resolution at hand of Ubiquitin in hyperpolarized water by a statistical analysis of time-series of 1D dDNP spectra.

References
1. P. Kaderavek, F. Ferrage, G. Bodenhausen and D. Kurzbach, Chem. Eur. J., 2018, 24, 13418-13423.
2. O. Szekely, G. L. Olsen, I. C. Felli and L. Frydman, Anal Chem, 2018, 90, 10, 6169-6177.
3. D. Kurzbach, E. Canet, A. G. Flamm, A. Jhajharia, E. M. Weber, R. Konrat and G. Bodenhausen, Angew Chem Int Ed Engl, 2017, 56, 389-392.
4. G. Olsen, E. Markhasin, O. Szekely, C. Bretschneider and L. Frydman, J Magn Reson, 2016, 264, 49-58.
5. G. L. Olsen, O. Szekely, B. Mateos, P. Kadeřávek, F. Ferrage, R. Konrat, R. Pierattelli, I. C. Felli, G. Bodenhausen, D. Kurzbach and L. Frydman, 2019, in preparation.

Speaker: Prof. Dennis Kurzbach (University of Vienna)
• 11:55
SABRE Chemistry and Spin Physics for High Precision Measurements and Biomedical Applications 35m

SABRE (Signal Amplification By Reversible Exchange) allows for rapid, affordable and repeated hyperpolarization of molecules directly in room temperature solutions. SABRE has many applications, ranging from biomedical to high precision measurements. To achieve the full potential, we investigated key steps in spin physics, chemistry, and engineering. Specifically, we (1) engineered membrane reactors for continuous polarization, (2) we created catalytic SABRE systems for hyperpolarization of a variety of drugs and water, and investigated hyperpolarization spin physics leading us to new SABRE and PHIP modalities with intriguing field dependence, (3) we efficiently polarized and detected heteronuclei at different magnetic fields and (4) we established low-field rare spin spectroscopy displaying masing effects, ideal for ultra-high precision measurements:

(1) High SABRE polarization was obtained under continuous flow using a membrane reactor for parahydrogen dissolution. This enables a continuous stream of hyperpolarized solution for injection or to feed a high precision Zeemann MASER.
(2) We hyperpolarized popular prescription drugs (e.g. anti-tumor, fungicides and antibiotics) and amino acids. Furthermore, we hyperpolarize water and find an atypical dependence on polarization transfer field, which speaks to a novel One-H polarization transfer mechanism.
(3) We also investigate heteronuclei such as 13C, 15N and 19F at high and low fields. In this context, we use the concept of LACs (level anti crossings) to advance SABRE-SHEATH and SLIC-SABRE approaches for heteronuclear hyperpolarization. A variety of novel targets with high hyperpolarization levels and long polarization decay time constant (T1, TS) will be presented.
(4) Finally, our advances enabled the parahydrogen fueled NMR RASER (radiowave amplification by stimulated emission of radiation). The continuous hyperpolarization pumps the RASER and multimode operation allows for ultra-high precision measurements in the micro-Hz regime and beyond.

All these aspects advance the SABRE method towards important applications in various areas of science including biomedical applications and high precision measurements.

Speaker: Dr Sören Lehmkuhl (North Carolina State University)
• 10:30 12:30
MRI Developments: Session 37 Lecture Hall B

Lecture Hall B

Henry Ford Building

• 10:30
Developments of NMR for Applications in Chemical Engineering and Medicine 35m

To enable wider application of NMR/MRI technologies in science, engineering and medicine, approaches must be specialised, accessible, simple and affordable. In this context this lecture will give recent examples from our journey of pushing MR boundaries.

We will report on the parallel acquisition of q-space, thus enabling real time monitoring of averaged propagators [1]. We will also discuss Magnetic Resonance Pore Imaging (MRPI) at resolutions well beyond the limits of conventional MRI [2] and demonstrate how recent advances in Rheo-NMR enable further insight into complex fluids under shear [3].

With respect to medical applications the lecture will report on measuring averaged fractional anisotropy [4], discuss options for the measurement of bone-to-total-volume ratios [5], metabolic rates of cells under mechanical stress and developments towards sensors for blood oxygenation [6].

To expand NMR and MRI into non-conventional areas as highlighted above it is necessary to develop affordable electronics [7] which meets the requirements of purpose built MR systems. Furthermore, experimental protocols and data analysis are required to be robust, especially under conditions of low signal to noise ratios [8,9]. Together with appropriate magnet systems we see great potential of NMR and MRI to be used in process control, quality assurance, point of care sensors as well as in academic and industrial research.

[1] Kittler, W. et al., Phys. Rev. E 92 023016 (2015).
[2] Hertel, S. A. et al., Phys. Rev. E 92 012808 (2015).
[3] Galvosas, P, et al., Magn. Reson. Chem.. Doi:/10.1002/mrc.4861 (2019).
[4] Zong, F. et al., Magn. Reson. Chem. 55 498-507 (2017).
[5] Brizi, L. et al., Magn. Reson. Med. 79 (2018).
[6] Thomas, D., Victoria University of Wellington, Hdl:123456789/3. Web. (2018).
[7] Ang, A. et al., Proceeding ISMRM-ESMRMB 16-21 June 2018 Paris, France.
[8] Teal, P. D. and C. Eccles, Inverse Problems 31 045010 (2015).
[9] Anjum, A. R. et al., Mag. Res. Chem. 56 740-747 (2018).

Speaker: Prof. Petrik Galvosas (MacDiamid Institute for Advanced Materials and Nanotechnology, School of Chemical and Physical Sciences, Victoria University Wellington)
• 11:05
New developments in production of proton-hyperpolarized propane gas for MRI 25m

Hyperpolarization allows one to increase the NMR sensitivity by several orders of magnitude. The main drivers behind the development of hyperpolarization techniques are their biomedical applications. For example, the inhalation of hyperpolarized noble gases, such as 129Xe and 3He, enables functional imaging of lung diseases. However, highly specialized 129Xe and 3He MR equipment and software is required which is not available on conventional clinical MRI scanners. Therefore, 1H-hyperpolarized gases, e.g. propane, represent a promising alternative. Hyperpolarization of propane can be accomplished by pairwise addition of parahydrogen to propylene over heterogeneous catalyst.
Here, we present our recent results on hyperpolarization of propane gas. We developed propane polarizer that enables production of hyperpolarized propane on a clinical scale (production rate >0.3 L just in 2 s, that is more than an order of magnitude greater than that demonstrated previously). Importantly, high polarization levels (~1%) can be retained despite the increase in production rate, allowing stopped-flow slice-selective high-resolution 2D MRI visualization. It was demonstrated that at ~0.05 T magnetic field hyperpolarized propane occurs as a long-lived spin state, which lifetime TLLS is ~3 times greater than T1. The use of buffering gases leads to the increase of propane polarization despite slight TLLS decrease. Cryocollection of hyperpolarized propane, which can be employed for buffering gas separation, increases TLLS up to 14.7 s in the liquid state, which is higher than that of gaseous hyperpolarized propane at any pressure studied. We also explored feasibility of propane hyperpolarization via hydrogenation of cyclopropane with parahydrogen. 1H polarization up to 2.4% was obtained, that is several times greater than that obtained with propylene as a precursor. The resulting NMR signal enhancement was sufficient for 2D MRI despite relatively low chemical conversion of cyclopropane substrate.
This work was supported by Russian Science Foundation (grant #17-73-20030) and DOD CDMRP W81XWH-15-1-0271.

Speaker: Dr Oleg G. Salnikov (International Tomography Center SB RAS and Novosibirsk State University)
• 11:30
Ultra-high field MRI and MRS: Opportunities and Challenges from Anatomical Imaging and Metabolite Detection for Biological Specimens 25m

Magnetic Resonance Imaging at ultra-high field strengths (17-22 T) provides both opportunities and challenges for non-invasive imaging of biological specimens. As low sensitivity is the most common drawback of MRI applications, the most apparent opportunity of ultra-high field imaging manifests itself by an augmentation in the Signal-to-Noise ratio (SNR). To this end, using similar RF coils, we found that the SNR increased by a factor of 6 when going from 14.1 T to 22.3 T. This SNR increase can be used for faster imaging (a factor of 32), higher resolution imaging reaching (5.5 µm)3[1] for an acquisition time of 58 h 35 min (3D FLASH, field-of-view 1.6 x 1.1 x 1.1 mm3), and direct metabolite detection by localized spectroscopy (10 mM acetate in a voxel volume of 27 nL in 18 min). In addition to spectroscopy, the increased chemical shift dispersion offered by ultra-high magnetic fields also benefits chemical exchange techniques such as Chemical Exchange Saturation Transfer (CEST) imaging which allows indirect metabolite detection by making use of the exchange of labile metabolite protons with water protons. We show that CEST at 17 T outperforms CEST at 7 T not only in terms of sensitivity but also by better separating the contributions of individual metabolites (glutamate vs. glucose). Lastly, concerning the challenges of MRI at ultra-high field, the increase of susceptibility artifacts caused by air spaces and paramagnetic ions is detrimental to image quality. We will show such effects and discuss possible solutions by examples of in vivo root specimens of M. truncatula and electrode materials.

[1] Krug et al., High spatial and temporal resolutions with increasing B0 and decreasing transceiver coil dimensions, (manuscript in preparation).

Speaker: Ms Julia R. Krug (Laboratory of BioNanoTechnology and Laboratory of Biophysics,Wageningen University & Research)
• 11:55
In vivo three-dimensional extracellular pH mapping of tumors using EPR 35m

Background and aim: Acidosis and low-oxygen status are hallmarks of solid tumors. Acidification in extracellular space in solid tumors reflects a shift of cellular metabolism for tumors. Thus, visualization of extracellular pH (pHe) is useful to understand the pathophysiological status of tumors. A method of three-dimensional (3D) pHe mapping of murine tumors using electron paramagnetic resonance (EPR) is introduced in this talk.
Methods: pHe was measured with a 750-MHz home-built continuous-wave EPR spectrometer/imager using a pH-sensitive nitroxyl radical probe (dR-SG) [1,2]. pHe maps were obtained by four-dimensional spectral-spatial EPR imaging. For the proof-of-concept experiment, solution samples, adjusted to 6.60, 6.80, and 7.00 pH units, were visualized. Murine squamous cell carcinoma (SCC VII) cells were implanted into the right hind legs of mice. Tumor-bearing mouse legs were monitored 5 and 8 days after the implantation. Also, tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells (MIA PaCa-2, SU.86.86, and Hs766t) were measured when the tumor volumes reached approximately 1 cm3.
Results: 3D pH maps were reconstructed for three tubes containing different pH solutions. The pH resolution was achieved at 0.078 pH units, and the trueness of pH values was 0.026 pH units [3]. 3D pHe maps of SCC VII tumors on day 5 and 8 exhibited the progress of acidification. The tumor xenograft mouse models exhibited different levels of acidification and inhomogeneous spatial distribution of pHe in tumor tissues.
Conclusion: In vivo pHe mapping was demonstrated with tumor mouse models. The results suggest that the method of 3D pH mapping can be applied to future studies of tumor mouse models that involve tumor acidification.

[1] A. A. Bobko et al., Magn. Reson. Med. 67, 1827 (2012).
[2] H. Sato-Akaba et al., Anal. Chem. 81, 7501 (2009).
[3] D. A. Komarov et al., Anal. Chem. 90, 13938 (2018).

Speaker: Prof. Hiroshi Hirata (Hokkaido University)
• 10:30 12:30
Solution-state NMR Methods: Session 40 Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 10:30
Investigating the dynamic conformational landscape of G-protein-coupled receptors (GPCRs) 35m

GPCRs belong to a family of ca. 850 plasma membrane-embedded proteins, which as molecular signalling switches control a wide range of physiological processes in health and disease. Activation of the individual receptor proteins is initiated via extracellular stimuli, such as photon uptake, or binding of small molecules, peptides, proteins, ions, lipids etc. This initial stimulus leads to conformational changes in the GPCR that favour intracellular binding partners to interact at the cytoplasmic receptor side. This then triggers a variety of downstream signalling cascades.

GPCRs represent the largest and most intensely studied class of drug targets and it is becoming increasingly clear that beyond the canonical activation through orthosteric ligands there is influence from many additional molecular factors, revealing the regulation of these receptors to be highly allosteric.

A wealth of structure data has provided a detailed static view of the basic conformational features of receptor activation. Yet, GPCRs are highly dynamic and adaptable entities that populate a complex and changing energy landscape. Not surprisingly therefore, the exact details of how signal propagation progresses from the extracellular side of the receptor to the cytoplasm are still unclear and the mechanistic determinants that allow a receptor to differentiate between the different interaction partners are still poorly understood.

Various NMR studies have provided experimental evidence on the dynamic nature of GPCRs. Here we investigate the class A receptor β1 adrenergic receptor (β1AR) using a range of complementary 13C and 19F NMR techniques that reveal the response of the ligand binding pocket as well as the cytoplasmic side of the receptor to probing with ligands, nanobodies and mini-G protein. Our studies show the receptor existing as a mixture of low populated intermediates that interconvert with each other, with populations as well as exchange kinetics influenced by ligand binding.

Speaker: Dr Daniel Nietlispach (University of Cambridge, Department of Biochemistry)
• 11:05
Light-induced changes in the conformational dynamics of a reversibly photo-switchable fluorescent protein revealed by solution NMR spectroscopy 25m

Reversibly photo-switchable fluorescent proteins (RSFPs) are important tools for microscopy and other biotechnological applications. They are currently routinely used for Near-Field Super Resolution Microscopy techniques, e.g. RESOLFT (1). Their characteristic switching between a fluorescent “on” state and a non-fluorescent “off” state, combined with signal processing algorithms has allowed to improve the resolution of cellular imaging down to of a few nanometers. Crystallographic studies of such RSFPs have provided crucial insights on their atomic structure, that has guided the field of fluorescent protein engineering in the search for better tags. However, the crystal forms of such proteins studied at cryogenic temperatures does not provide a realistic picture of the conformational dynamics, and how they influence the photophysics of the RSFP. So far, only a single NMR study for the RSFP Dronpa has been reported in the literature (2). Here, we present a comprehensive NMR study of rsFolder, a green negative RSFP (3). Using a portable in-situ laser illumination device, we were able to perform NMR assignments and derive local dynamic information for both, the fluorescent “on” and “off” states of rsFolder, aided by Laser-driven Exchange NMR experiments. After photo-switching, rsFolder experiences significantly enhanced millisecond time scale dynamics, affecting mostly the environment of the chromophore as detected by extensive line broadening of backbone and side chain resonances. H/D exchange measurements also revealed a global destabilization of the β-barrel region facing the phenol ring of the endogenous chromophore, as well as the chromophore-connecting helical structures. Single-point mutants of rsFolder have been used in an attempt to make correlations between the NMR-observed features and the different photophysical properties of these mutant RSFPs.
(1) Hell, S.W., 2003. Nat.Biotechnol. 21, 1347–1355.
(2) Mizuno, H. et al, 2010. J.Biomol.NMR. 48, 237-246.
(3) El Khatib, M. et al, 2016. Sci.Reports. 6:18459(1-12)

Speaker: Ms Nina-Eleni Christou (Institute de Biologie Structurale, UGA, CNRS, CEA)
• 11:30
Ribosome induced riboswitch structure melting 25m

A large ribonucleoprotein complex called the ribosome is responsible for several steps of protein synthesis in all organisms. In bacteria, regulation of translation begins at initiation. Despite the availability of structural information, we still lack a clear understanding of how the ribosome encounters folded mRNA structures during translation initiation.
A general mechanism for initiation could involve a ribosome standby binding to single-stranded regions of the mRNA in the vicinity of the ribosome binding site, representing the first docking step and thus providing a general mechanism for initiation. This standby model describes the paradox of high translation rates from highly structured mRNAs with a sequestered RBS. Until now, little attention has been paid to standby complexes, particularly those of structured mRNAs.
Riboswitches are structured cis-acting RNA elements located in the 5’-untranslated region of mRNA that regulate gene-expression at the level of transcription, RNA cleavage and translation in response to binding of a cognate ligand.
The direct regulation by riboswitches operates by interfering with the formation of translation initiation complexes.
However, a structural description of the interaction between riboswitches and ribosomes during translation initiation is currently missing. It therefore has remained elusive how further structured elements can be fully accommodated within the initiation complex. We investigate the influence of the 30S ribosome on the function of the adenine-sensing riboswitch from Vibrio vulnificus by NMR spectroscopy. Surprisingly, the adenine-induced allosteric switch leading to an opening of the RBS is insufficient for efficient translation initiation. Additional stable structured elements around the initiation region prevent mRNA accommodation in the ribosome decoding channel.
Our results show that the full activity of the riboswitch is only obtained upon concerted interaction with adenine and ribosomal proteins. The RNA chaperone activity of the ribosomal protein S1 is needed for melting of secondary structures that would otherwise preclude complex formation.

Speaker: Mrs Vanessa de Jesus (Goethe University Frankfurt am Main)
• 11:55
Leveraging the Achilles heel of high-gamma nuclei to observe low-gamma nuclei. 35m

Sensitivity and resolution have been the two important traits in NMR of biomolecules. With the advent of cryogenically cooled probed and non-uniform sampling methods, the battle of sensitivity and resolution has to be revisited. 1H has long enjoyed the limelight due to its inherent sensitivity. The large gyromagnetic ratio of 1H it is nemesis when dealing high molecular weight systems since the dipolar contribution to relaxation is governed by the square of the gyromagnetic ratio. However, low gamma nuclei, though insensitive, have slower relaxation rates thus providing sharper resonances – a desired factor when dealing with a crowded spectrum. With reduced dipole-induced relaxation, the relaxation of the low gamma nuclei is often affected by chemical shift anisotropy (CSA). Here, we follow on the previously established TROSY effect-which cancels part of the CSA induced relaxation of the low-gamma nuclei using the dipole of the high-gamma nuclei- and develop 13C and 15N detected experiments for large molecular weight systems. The architecture, unique advantages and the limitations of these experiments will be presented.

Speaker: Dr Haribabu Arthanari (Havard Medical School)
• 10:30 12:30
Spin Physics: Session 36 Lecture Hall C

Lecture Hall C

Henry Ford Building

• 10:30
Ultrasensitive beta-detected NMR at CERN: first results in physics and biology 35m

Beta-detected NMR is up to 10 orders of magnitude more sensitive than conventional NMR, because it is based on the detection of beta-particles from hyperpolarized short-lived nuclei. Our project aims at applying it for the first time to liquid samples relevant in chemistry and biology, thus extending its use from nuclear structure and material science studies in solid environments.
Our experimental setup, built in 2016 and upgraded in 2017 and 2018, is located at the CERN-ISOLDE facility, where over 1000 different radioactive nuclei can be produced. We use optical pumping with lasers on isotopes of different metallic elements, resulting in nuclear polarizations up to 90%. The decrease in the anisotropic emission of beta radiation from such nuclei is then used to detect the NMR response, leading to the extreme sensitivity of beta-NMR.
First studies on 26Na in liquid samples were performed by us in 2017, which lead to much narrower beta-NMR resonances than seen previously in solid hosts. Thanks to the shimming and active stabilisation of our 1.2 T electromagnet at the 1 ppm level, in 2018 we could determine the magnetic moments of several short-lived Na nuclei with about 100 times improved precision. This provides a self-consistent set of nuclei to be used in beta-NMR studies in liquid samples, connected to the moment of stable 23Na. In 2018 we furthermore probed the interaction of Na cations with DNA G-quadruplex stuructures, present e.g. in telomeres. Present upgrades to the experimental setup should allow to apply this approach to isotopes of more chemical elements (e.g. K, Cu or Zn) in an even broader range of research topics, such as alkali-metal batteries.

This contribution will introduce the technique and describe the experimental setup, and will concentrate on the most recent results in physics and biology, followed by a short outlook.

Speaker: Prof. Magdalena Kowalska (UNIGE, CERN)
• 11:05
Direct magnetic field dependence of NMR shielding 25m

Nuclear shielding is considered independent of the magnetic field strength when analysing NMR experiments. However, already in 1970, Ramsey proposed on theoretical grounds that this may not be valid for heavy nuclei. Here we present experimental evidence for the direct field dependence of shielding, using 59Co shielding in Co(acac)3 dissolved into chloroform as an example. This low-spin diamagnetic Co(III) complex features a very large and negative nuclear shielding constant of the central Co nucleus. We carry out variable temperature NMR experiments in four different field strengths ranging from 7.05 to 18.79 T. As there is a well-known sensitivity of the 59Co NMR frequency to temperature, we introduce Xenon gas into the sample and use the known temperature dependence of the 129Xe signal to calibrate the temperature. Signal from a Xenon gas sample is used as a frequency reference. The experiments result in temperature dependent magnetic field dependence in the order of -10-3 ppm T-2 for the 59Co shielding constant, arising from the direct modification of the electron cloud of the complex by the field. First-principles non-linear response theory results in values ranging from -10-5 to -10-3, in reasonable agreement with the experiment. Upon increasing field strengths available in contemporary NMR setups, the direct magnetic field dependence of NMR parameters becomes a factor to take into account in studies of materials and molecular structures. Furthermore, direct field-induced effects may in the future provide entirely new tools for materials characterisation.

Speaker: Dr Anu M. Kantola (University of Oulu)
• 11:30
Towards single-shot readout of NV centers in diamond by low-temperature spin-to-charge conversion 25m

We present our recent progress in implementing an improved readout scheme for the nitrogen-vacancy (NV) center's spin-state combining resonant excitation at low (4 Kelvin) temperature with spin-to-charge conversion. Resonant excitation exploits that the optical excitation spectrum at low temperature has sufficiently narrow linewidths[1,2] to selectively address the spin-sublevels. In combination with a second laser pulse, a spin-to-charge conversion[3,4] protocol can be implemented, where the NV center is spin-selectively excited and converted to different charge-states. These are more stable than the initial spin-state and can be read-out with single-shot fidelity, even by inefficient collection optics.

Compared to the state-of-the-art readout[5], this work promises to accelerate readout by a factor of up to 100. Besides, laser power in the optical regime can be reduced, which lowers the risk of photodamage for future sensing experiments with biological samples.
We expect our scheme to become a core enabling tool for single-molecule electron spin resonance detected by NV centers.

References

[1] A. Batalov, Physical Review Letters 102, 195506 (2009)
[2] M.W. Doherty, New Journal of Physics 13, 025019 (2011)
[3] B.J. Shields, Physical Review Letters 114, 136402 (2015)
[4] X.-D. Chen, Physical Review A 7, 014008 (2017)
[5] D.A. Hopper, Micromachines 9, 437 (2018)

This project has received support from DFG via projects SPP1601 and RE3606/3-1.

Speaker: Dr Friedemann Reinhard (TU München, Walter Schottky Institut)
• 11:55
Magnetic Resonance Spectroscopy of A Single Molecule 35m

Magnetic resonance (MR) is one of the most important techniques for characterizing compositions, structure and dynamics of molecules. Over the past several years, quantum sensing with Nitrogen-Vacancy (NV) center has opened a new door for magnetic resonance spectroscopy of a single molecule. In my talk, I will mainly introduce several new experimental results on both of methods and biology applications. (I) Zero-field electron spin resonance (ESR) spectroscopy on nanoscale. We successfully measured the zero-field ESR spectrum of a few electron spins, by precisely tune the energy levels of NV centers to be resonant with the target spins, and directly resolved the hyperfine coupling constant. This work break the sensitivity limitation and open the door of practical applications of the zero-field ESR. (II) ESR spectroscopy of a single protein in poly-lysine and a single DNP duplex in aqueous solution. The work represents a step forward towards magnetic resonance investigation of biomolecules in their native environments at the single-molecule level. (III) We realized one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy of two coupled nuclear spins and resolved its structure.

Reference
[1] Fazhan Shi, et al., Single-DNA electron spin resonance spectroscopy in aqueous solutions, Nature Methods 15, 697 (2018)
[2] Fei Kong, et al., Zero-field electron spin resonance spectroscopy on na-noscale, Nature Communications 9, 1563 (2018)
[3] Fazhan Shi, et al., Single-protein spin resonance spectroscopy under ambient conditions, Science 347, 1135 (2015)
[4] Fazhan Shi, et al., Sensing and atomic-scale structure analysis of single nuclear spin clusters in diamond, Nature Physics, 10, 21-25 (2014)
[5] Tobias Staudacher, et al., Nuclear magnetic resonance spectroscopy on a (5nm)3 volume of liquid and solid samples, Science, 339, 561 (2013)
[6] Zhiping Yang, et al., Two-dimensional nanoscale nuclear magnetic resonance spectroscopy enhanced by artificial intelligence, arXiv:1902.05676 (2019)

Speaker: Prof. Fazhan Shi (University of Science and Technology of China)
• 12:30 13:30
Brunch 1h
• 13:30 14:10
Awards Session Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

Suraj Manrao Student Poster Awards, FEBS Journal Poste Awards, Wiley Awards, JMR Awards

• 14:10 15:30
Plenary Lectures Max Kade Auditorium

Max Kade Auditorium

Henry Ford Building

• 14:10
Nanoscale magnetic spin resonance using the nitrogen vacancy centre in diamond 40m

The nitrogen-vacancy (NV) centre in diamond is an optically addressable single spin-1 electronic system with relatively long coherence persisting at room temperature, making it ideal for a range of nanoscale quantum sensing applications. The NV system is sensitive to local magnetic and electric fields, as well as material properties such as strain and temperature. An exciting direction is the development of the NV centre as a quantum probe for nanoscopic magnetic resonance applications. In this talk, we will briefly review the NV centre and some of the seminal sensing and imaging demonstrations, and focus on new results based on controlled cross-relaxation between the NV quantum probe and target electron and nuclear spin systems. Experimental demonstrations of both electron and nuclear spin resonance using these techniques will be described, including applications in chemical detection, bio-magnetic characterisation, and hyperpolarisation of nuclear spins external to the diamond substrate. Finally, we will look at ideas for using such controllable quantum spin probes in bio-molecular structure determination.

Speaker: Prof. Lloyd C. L. Hollenberg (School of Physics, University of Melbourne, Australia)
• 14:50
Dynamic Complexes and Complex Dynamics - NMR Studies of Large Scale Protein Motions 40m

Proteins are inherently dynamic, exhibiting conformational freedom on many timescales,1 implicating structural rearrangements that play a major role in molecular interaction, thermodynamic stability and biological function. Intrinsically disordered proteins (IDPs) represent extreme examples where flexibility defines molecular function. IDPs exhibit highly heterogeneous local and long-range structural and dynamic propensities, sampling a much flatter energy landscape than their folded counterparts, allowing inter-conversion between a quasi-continuum of accessible conformations.2 We are combining multifield NMR relaxation measurements and ensemble MD approaches to develop a unified description of the dynamics of IDPs as a function of environmental conditions.3-5
In spite of the ubiquitous presence of IDPs throughout biology, the molecular mechanisms regulating their interactions with physiological partners remain poorly understood. We use NMR spectroscopy to map these complex molecular recognition trajectories at atomic resolution, from the highly dynamic free-state equilibrium to the bound state ensemble. Examples include the replication machinery of Measles virus, where the highly (>70%) disordered phosphoprotein initiates transcription and replication exploiting weak interactions with ordered and disordered domains of the nucleoprotein,6,7 the nuclear pore, where weak interactions between the nuclear transporter and highly flexible chains containing multiple ultra-short recognition motifs, facilitate fast passage into the nucleus.8 and large-scale domain dynamics in Influenza H5N1 polymerase are essential for import into the nucleus of the infected cell.9
[1]. Lewandowski et al Science 348, 578 (2015)
[2]. Jensen et al Chem Rev 114, 6632 (2014)
[3]. Abyzov et al J.A.C.S. 138, 6240 (2016)
[4]. Salvi et al Angew Chem Int Ed. 56, 14020 (2017)
[5]. Salvi et al Science Advances In Press (2019)
[6]. Schneider et al J.A.C.S. 137,1220 (2015)
[8]. Milles et al Science Advances 163, 734 (2018)
[8]. Milles et al Cell 112, 3409 (2015)
[9]. Delaforge et al J.A.C.S. 137 (2015)

Speaker: Dr Martin Blackledge (Protein Dynamics and Flexibility by NMR)
• 15:30 15:50
Closing Remarks 20m Max Kade Auditorium