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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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,...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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),...
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...
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...
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...
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...
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...
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]...
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:...
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,...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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,...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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,...
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...
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...
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...
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,...
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...
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...
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...
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...
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]. ...
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...
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....
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...
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...
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...
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...
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...
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,...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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)...
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...
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 ...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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,...
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...
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...
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...
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:...
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...
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...
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...
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...
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...
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...
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...
Spin-diffusion (SD) was amongst the first methods proposed to spatially transfer polarization between dipolar-coupled nuclear spins. Lab-frame proton spin-diffusion (PSD) has proved particularly useful in structural characterization of a large variety of molecules. Perturbation theory treatment of the SD phenomenon states that during SD, the rate constant of magnetization exchange between two...
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...
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...
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,...
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...
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...
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]...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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....
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
In contrast to most proteins, RNAs can adopt more than one conformational state of similar stability. The different stable conformations are directly linked to the RNA’s biological function. Examples for such functional multi-state RNAs are riboswitches, ribozymes and RNA thermometers, where either external factors such as small molecular metabolites and temperature or the intrinsic...
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...
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...
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,...
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...
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...
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...
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...
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...
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...
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...
The application of Diffusion Ordered NMR Spectroscopy has vastly increased in the last years, which illustrates the benefits of monitoring diffusion in solution NMR. Consequently much effort has been ventured to improve the application of diffusion measurements in NMR [1]. In this work we introduce a pulse sequence that enables the spectroscopist to perform DOSY experiments in a matter of tens...
Elena Shanina, Sakonwan Kuhaudomlarp‡, Priscila Gomes†, Eike Siebsƒ, Alexander Titzƒ, Didier Rognan†, Anne Imberty‡, and Christoph Rademacher
Max Planck Institute of Colloids and Interfaces, Potsdam, Germany;
‡ Univ. Grenoble Alpes, CNRS, CERMAV, Grenoble,...
129Xe is classically hyperpolarized using the well-established spin exchange optical pumping (SEOP) method. At optimal conditions and using 129Xe enriched gas, SEOP can provide up to 3.6L/h of xenon hyperpolarized to 30%, enabling routine clinical 129Xe MRI [1]. Besides SEOP, sublimation-DNP was proposed as an alternative technique to produce hyperpolarized...
Nanodiamonds (NDs) are a subject of increased application interest based on the possibilities of their use in medical imaging [1] and as drug delivery systems [2, 3]. Fluorescent NDs are addressed as they emit single-photon luminescence due to a number of optically sensitive paramagnetic defects, particularly nitrogen-vacancy centres. The knowledge on how the defect types and distribution in...
Signal Amplification By Reversible Exchange (SABRE) is a method based on the transfer of parahydrogen (p-H2) polarization to the substrate nuclei due to the constant exchange of p-H2 and ligands over metallic Ir center of metal-organic complex. Generated levels of polarization provide the higher signal of the ligand. In the experiments, when SABRE process occurs in high...
In this contribution, we present a simple analytical fitting approach based upon a power-law model of the segmental orientation autocorrelation function (OACF), by the way of which an effective power-law time scaling exponent and the amplitude of the OACF can be estimated from multiple-quantum (MQ) NMR data at any given temperature [1]. This obviates the use of the time-temperature...
Chloride salts have wide applications in pharmaceuticals, whether they are used for isolating and purifying the active pharmaceutical ingredients, or for providing more stability and/or solubility to the drugs. 35Cl nucleus has a 75% natural abundance and it is quadrupolar, with a nuclear spin I=3/2. In many organic compounds where it is covalently bound, the 35Cl nucleus...
Double Quantum NMR is a well-established method to study spatial proximities and local molecular dynamics in solid samples. In many cases, more or less restricted local dynamics of polymer chains has been analyzed. Here we studied the local packing of phosphate groups during the crystallization of precise polyethylene obtained from acrylic diene metathesis (ADMET) polycondensation with...
Most materials are heterogeneous on the millimeter resolution of magnetic resonance imaging (MRI) in the sense that they are comprised of multiple microscopic domains with different chemical and diffusional properties. Consequently, the measured MRI signal on the voxel scale is highly unspecific as it originates from the average of the signals arising from these domains. However,...
An unexpected great challenge in dDNP [1] has recently emerged with the use of ultra-shielded DNP magnets; the preservation of hyperpolarization during transfer from one ultra-shielded magnet to the other in ultra-low field regions, as magnetization not only relaxes but can be wiped-out in a non-adiabatic process.
We have recently proposed the design of a fluid path for dDNP [2] based on a...
Short-lived and low-abundance enzyme intermediate complexes represent a mainstream of transient protein complexes in enzyme catalysis and signal transductions. Many transient and unstable protein intermediate complexes are generated in real-time reactions or non-equilibrium conditions, however, resonance assignments of these unstable complexes are a challenge and structural determination of...
The automation of NMR structure determination remains a significant bottleneck towards increasing the throughput and accessibility of NMR as a structural biology tool to study proteins. The chief barrier currently is that obtaining NMR assignments at sufficient levels of completeness to accurately define the structures by conventional methods requires a significant amount of spectrometer time...
It is known that cobalt exhibits polymorphism: it exists in both hcp and fcc phases. Synthesis method and other thermodynamic conditions are known to play a role in determining the phase composition of cobalt nanoparticles. In this work, we have studied the phase composition of the cobalt nanoparticles synthesized using two different solvents (water) and ethanol. XRD measurements confirm the...
NMR is a primary method of measurement that provides quantitative results without the need of a standard of the same measurand. Amongst different procedures for quantification of organic compounds by NMR, the International Bureau of Weights and Measures (BIPM) encourages the use of internal standard (IS) NMR as it presents less sources of errors and smaller uncertainties. The use of a...
NMR spectroscopy is an efficient method for obtaining quantitative data on the fractional 13C enrichment of metabolic intermediates or end-products. In biosynthetically directed fractional 13C labelling using uniformly 13C labelled carbon source, the biomass becomes 13C labelled in a metabolic flux distribution dependent manner and the cleavage and...
Exchange processes with the solvent may reveal useful information about intrinsically disordered proteins and their interactions. We introduce a new method to evaluate amide proton exchange rates that relies on the creation of a triple spin order state, i.e. 4CzNzHz. This spin state is left free to evolve in a variable mixing time during which it undergoes...
Radio-frequency signals can be up-converted to the optical regime through electromechanical and optomechanical couplings [1]. Applying this technique, we reported on proof-of-principle demonstrations of Electro-Mechano-Optical (EMO) NMR [2, 3]. Towards realization of practical EMO NMR, we present here the design and fabrication of a compact rf-to-light transducer that can be installed inside...
The large diversity of nucleic acid structures can be expanded even further by the introduction of metal-mediated base pairs in which base pairs are generated by coordination to metal ions instead or in addition to hydrogen bonds.[1,2] These metal-mediated base pairs consist of either natural or artificial nucleobases and are frequently used for the programmable and site-specific insertion of...
Direct interaction between intrinsically disordered proteins is often difficult to be characterized hampering the elucidation of their binding mechanism. Particularly challenging are the cases of extreme fuzziness of the complex, which is now appearing as a common interaction mode,requiring new models for their description.1
So far, nuclear magnetic resonance spectroscopy has...
2D J-resolved NMR spectroscopy, separating chemical shifts and J couplings along two independent dimensions, serves as a promising tool for studying molecular structures and compositions. In practice, the classic homo-nuclear 2D J-resolved spectroscopy [1], generally suffers from phase-twist lineshape and field inhomogeneity effects, thus rendering its potential applications limited. Here, a...
NMR spectroscopy is a nondestructive characterization technique that provides detailed information on molecular structures with incomparable specificity. With NMR, it is possible to observe chemical reactions, detect chemical substances, and to determine concentrations.
Commercial NMR devices are usually bulky, expensive and power hungry. To change this situation, our approach uses integrated...
Dissolution dynamic nuclear polarization (D-DNP) is currently the subject of many new developments in view of boosting the sensitivity of nuclear magnetic resonance spectroscopy (NMR) and imaging (MRI). Most D-DNP probes are designed for one or two nuclei at most. The investigation of multiple nuclei, therefore, requires manufacturing a number of different costly probes. In addition, changing...
Electron-nuclear double resonance (ENDOR) spectroscopy is the method of choice for detecting magnetic nuclei in biomolecules which contain an unpaired electron spin is. However, due to its low gyromagnetic ratio, the detection of deuterium couplings is a particular challenge for ENDOR spectroscopy. The standard Mims ENDOR sequence, generally used for small couplings, suffers from line shape...
α1-adrenoceptors (α1-ARs) comprise three G protein-coupled receptor (GPCRs) subtypes that stimulate smooth muscle contraction in response to binding adrenaline and noradrenaline. α1A-AR and α1B-AR are clinically targeted for treating hypertension and benign prostatic hyperplasia but are putative drug targets for neurodegenerative diseases. New subtype-selective tool compounds are required to...
Fitting a weighted sum of exponentials to a noisy exponential decay is an ill-posed problem. In the T2 relaxation context, it leads to non-unique solutions because different sets of relaxation times and amplitudes give identical least-square distance between the model and the experimental data. Conventional data analysis combine a non-negative least squares (NNLS) algorithm...
In modern organic and medicinal chemistry, fluorine is commonly used to enhance the chemical properties of molecules in many desirable ways: it may delay the metabolism of the molecule, reduce the toxicity of aromatic groups, or increase the bioavailability. As a result, it is estimated that more than 20% of commercial pharmaceutical APIs and 30% of agrochemicals contain at least one fluorine...
The hyperpolarization of biologically relevant molecules for signal-enhanced magnetic resonance detection holds great promise for studying biological processes in vitro and in vivo. Being important metabolites as well as the building blocks of peptides, the canonical amino acids are promising targets for hyperpolarization, and a number of parahydrogen based strategies have been...
Electron Paramagnetic Resonance (EPR) in single crystals is used to study paramagnetic intermediates at the active-site of an enzyme to fully resolve the magnitudes and orientations of the g-, hyperfine, and quadrupole tensors. The electronic structure, when combined with X-ray crystallography, is related to a proposed molecular geometry to gain a better understanding of the catalytic...
NaK and other non-selective channels are able to conduct both sodium (Na+) and potassium (K+) with equally high efficiency. In contrast to previous crystallographic results, we show that the selectivity filter (SF) of NaK in native-like lipid membranes adopts two distinct conformations that are stabilized by either Na+ or K+ ions. The atomic...
There exists a number of experimental methods which provide homodecoupled (pure-shift) 1H NMR spectra. Standard techniques (like PSYCHE [2] and TSE-PSYCHE [3]) require to record spectra in a pseudo-2D mode where chunks of data are acquired as separate FIDs. However, this often leads to very time-consuming experiments [1]. The problem with time rises, when a series of pure-shift proton spectra...
Pure-shift NMR [1] is a branch of techniques allowing the suppression of homonuclear J-couplings in an NMR spectrum. This gives a boost in resolution as multiplets in a spectrum collapse into singlets. The techniques rely on echo-type pulse sequences with selective pulses. In between the pulses, a short chunk of an FID is acquired. The whole FID is then constructed from these chunks.
The type...
The combination of a phase-modulated (PM) saturation pulse and symmetry-based dipolar recoupling into a rotational-echo saturation-pulse double-resonance (RESPDOR) sequence has been employed to measure 1H-14N distances. Such a measurement is challenging owing to the quadrupolar interaction of 14N nucleus and the intense 1H-1H homonuclear...
Magic‐angle spinning (MAS) is an essential ingredient in a wide variety of solid‐state NMR experiments. The standard procedures to adjust the rotor angle are not highly accurate, resulting in a slight misadjustment of the rotor from the magic angle (~54.74°) on the order of a few millidegrees. This small missetting has no significant impact on the overall spectral resolution, but is sufficient...
Development of new ESR instrumentation is an important priority of the Cornell University National Biomedical Center for Advanced ESR Technology (ACERT) at Cornell University. The ACERT high power (1.2kW) 95 GHz broadband spectrometer1 is currently the only system available to the scientific community allowing for 2D-ELDOR studies on biologically relevant aqueous samples at room temperature....
Unambiguous characterization of different acid sites in zeolites is of great importance in heterogeneous catalysis. In addition to various well-characterized extra-framework Al species, tri-coordinated framework aluminum species can also serve as Lewis acid site (LAS) in zeolites, which is however “NMR-invisible” due to its extremely distorted/asymmetrical local environment resulting in the...
The zeolites are well known for the decades for their excellent properties as adsorbents and catalysts. The large-scale application of these materials is ensured by petroleum chemistry as a part of fuel cracking catalysts (FCC) for gasoline production and hydrockacking catalysts (HCK) for diesel due to their thermal stability, strong acidity, and developed specific surface area. A main...
In the SWAMP effect (Surface Waters Are Magnetized by Parahydrogen), hyperpolarization of the protons of liquid water, methanol or ethanol is mediated by mesoporous silica-encapsulated Pt3Sn intermetallic nanoparticles (iNPs).1 Hydroxy protons exhibit stimulated emission NMR signals after a few seconds of bubbling of para-enriched H2 through a suspension of the...
Ultralow-field (ULF) nuclear magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) are promising methods allowing for, e.g., the simultaneous detection of multiple nuclei or imaging in the vicinity of metals. To overcome the inherently low signal-to-noise ratio that usually hampers a wider application, we present an alternative approach to prepolarized ULF MRS, employing...
Conventional methods for determining crosslink densities (CLDs) in elastomers, such as equilibrium swelling of vulcanizates (using the Flory-Rehner equation), and stress-strain measurements (using the Mooney-Rivlin equation) lack phase-specific distinction of crosslink densities in elastomer blends [1]. NMR spectroscopy offers better solutions to overcome these limitations, but studies...
The aggregation of beta-amyloid peptides is closely associated with Alzheimer's disease. We have used different chemical devices such as liposomes and reverse micelles to encapsulate the 40-residue beta-amyloid peptides so that the spatial confinement could lead to the formation of nonfibrillar aggregates of beta-amyloid peptides. These on-pathway beta-sheet intermediates were used to seed the...
The general problem of NMR and MRI is their low sensitivity. The NMR/MRI signal intensity is proportional to the nuclear spin polarization that is on the order of 10−5 or less at a common magnetic field of NMR/MRI. On the other hand, triplet dynamic nuclear polarization (triplet-DNP), a method to enhance NMR/MRI sensitivity using photoexcited triplet electrons, has the great...
The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H Ras, N Ras, and K Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best...
Abnormal protein aggregation is a common hallmark of Alzheimer’s disease and other neurodegenerative disorders. Despite the growing interest in their pathogenesis, no existing method can capture aggregate nucleation and subsequent growth at atomic resolution in real time. In this study, we have recently established high-sensitivity Rheo-NMR spectroscopy that enables us to detect atomic-level...
Recently the phenomenon of Liquid-Liquid Phase Separation (LLPS) has gained great interest among the scientific community given its participation in the formation of membraneless organelles that play crucial roles in the cellular environment 1. Great efforts are currently devoted to its structural and functional characterization. However, obtaining structural information at the molecular level...
Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes and act at the heart of fundamental cellular processes such as cell cycle control and signal transduction. The aberrant function of IDPs is therefore associated with severe diseases such as cancers and neurodegenerative diseases. Protein functions are mediated via motions, therefore a detailed characterization of...
Tc toxins are 1.7 MDa protein complexes that are found in insect- and human-pathogenic bacteria. After endocytosis, Tc penetrates the membrane of the host’s cells and translocates a deadly enzyme into the cytosol. The complex consists of three subunits: the 1.4 MDa TcA pentamer, which mediates target cell association, membrane insertion and toxin translocation, and two smaller subunits, TcB...
The inversion of NMR relaxation time is very important to study object’s molecular dynamics. The inversion is to solve the Fredholm integral equation of the first kind with non-negative constraints, which is known as an ill-posed problem. In this paper, a novel method is presented for NMR inversion based on the regularization method. The proposed objective function can transform the...
The ability to select the signal passing through chosen coherence pathways is a powerful feature of NMR. Nuclear singlet states are coherent states that have effective spin 0[1]. Singlet states can be used to sustain hyperpolarized signal [2], to study slow dynamic phenomena[3] and to filter signal originating from specific spin ½ pairs[4,5].
Despite being NMR silent, singlet states can be...
Motivation
Among other factors, the sensitivity of Magnetic Resonance Imaging (MRI) is limited by the low polarization of the sample investigated. Enhancing the polarization offers several orders of magnitude in signal enhancement. In comparison to e.g. Dynamic Nuclear Polarization (DNP), para-hydrogen (pH2) based approaches are less cost intensive, scale well and offer high...
Chocolate is obtained from cocoa bean (Theobroma Cocoa), which is previously fermented by microbial activity transforming it reaches until characteristic odour and flavour. This process is developed in three stages: 1st) Alcoholic fermentation, whereby sugars of husk and bean are consumed (first 48 h, aprox), 2nd) Acidogenic fermentation, where the alcohol yielded is consumed and it produces...
Selective anion binding to a catalytic host is a promising topic on the route to develop systems triggering catalytic activity. In this regard, cooperative hydrogen bonding by multiple 1,2,3-triazoles establishes a suitable environment for different anions. On this basis, the asymmetric dearomatization of quinolines was successfully introduced by anion binding catalysis [1]. However, the...
Oxidative stability of oils is an important quality issue especially during deep-fat frying process. It is well known that lipid oxidation can lead to changes in functional, sensory, and nutritive values and even the safety of fried foods. Extracts of many plants have been reported to have varying degrees of antioxidant activities, which increase the oxidative stability of fats and oils....
Multiple-quantum MAS (MQMAS) NMR spectroscopy is one of the most widely used techniques in solid-state NMR for gaining site-specific resolution of overlapping central-transition lineshapes for half-integer quadrupolar nuclei.[1,2] However, a major drawback of this method is the low MQ excitation and reconversion efficiency, which has led to broad variety of MQMAS sequences with improved...
Proteins in biological environments exist as part of crowded mixtures. Biopharmaceutical proteins for injections also often have to be formulated at very high concentrations (100-400 mg/mL) and as mixtures. Characterizing protein self-interactions vs cross-interactions in such complex systems in situ is challenging, but is required for adequate system description.
Recently our group and...
Glycosylation and spontaneous modifications are critical quality attributes of protein therapeutics that can impact drug efficacy and safety. Therefore they need to be tightly monitored during production, formulation and storage. Mass spectrometry and fluorescence-detected chromatography are currently the dominating methods for the analysis of post-translational modifications (PTMs) including...
The functions of lubricant base oil are largely correlated to its structure and composition, which are however very difficult to be measured simultaneously by current analytical methods. Mass spectroscopy could get quantitative group composition of base oil, but it is deficient in characterization of structures with branches. On the other hand, conventional NMR method is not only suffered from...
The aim of a metabolic study is the differentiation of biological populations by their metabolite composition. Even in a simple model the assessment of large numbers of samples is mandatory to produce statistically meaningful data. This requirement becomes even larger in the analysis of metabolite progression, for example in disease development, or mixtures of commodities, for example the...
Pulmonary compliance is an important index for evaluating the function of the lung, which could reflect the pulmonary ability to stretch and expand. Hyperpolarized 129Xe MR is a powerful tool in quantifying the microstructure and function of the lung in vivo, and it has unique advantages in measuring the gas volume in the lung. In this study, a proof-of-concept method was proposed...
The restrictive limits of molecular tumbling have made the study of large proteins and protein complexes a subject ideally suited to the strengths of magic-angle-spinning (MAS) solid-state NMR. Protons form a dense network of strong dipolar interactions and their shifts are sensitive reporters of protein structure and interactions. Higher MAS rates and highly deuterated samples have allowed...
Inorganic materials and compounds with transition metals find numerous applications within catalysis, environmental remediation as well as for energy storage and conversion due to their flexible oxidation states. However, their characterization is challenging due to the absence of long-range order, structural defects and/or the multi-phase nature of such materials. Solid state NMR (SSNMR) is...
The development of improved heterogeneous catalysts can be undertaken in a rational way by a better understanding of their atomic-level structures. Solid-state NMR spectroscopy is very well suited to the study of heterogeneous catalysts because it can give information on the local structure. However, the lack of sensitivity and resolution limits the characterization of surface sites, notably...
Ubiquitination and proteasome-dependent degradation of proteins is a major regulatory mechanism of protein function in eukaryotic cells[1]. The covalent attachment of ubiquitin to substrates occurs through the recruitment of three enzymes: a ubiquitin-activating enzyme E1, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin ligase. The E2 and E3 enzymes are the key components in...
Benchtop NMR spectrometers are a promising tool for chemical laboratories worldwide because of their moderate price and low handling cost. The easy setup of flow measurements makes them convenient to use for reaction/process monitoring. However, their applications are mainly limited to simple tasks. This is due to the insufficient spectral resolution and sensitivity, which are a consequence of...
The protein P2 is a member of the class of FABPs (Fatty Acid Binding Proteins). Its function in the organism is totransport fatty acids (FAs) out of the intercellular system and bring them to the myelin sheath around axons in the PNS (peripheral nervous system).
A reversible neuronal disease known as “Charcot-Marie-Thooth disease” leads to a reversible myelindegeneration and one theory...
Gadolinium is the most promising spin type for in cell studies of spin-labeled proteins. However, the availability of other bioresistant spin labels could aid the development of structural studies of biomolecules in a cellular context. The relatively small and flexible nitroxide label could offer many advantages in this area of research. We characterized iodoacetamide- and...
Block copolymers (BCP) due to their unique self-assembling properties represent important group among synthetic materials and they are believed to play significant role in future nanolithography. It appears that below certain critical temperature TODT (order-disorder transition temperature) two dissimilar subchains (blocks) become immiscible and tend to separate one from another gathering with...
BReast CAncer susceptibility protein 2 (BRCA2) is a 3000 residues long protein that serves as a plateform for proteins involved in genomic stability and mitosis. These functions are regulated by kinases phosphorylating BRCA2 unfolded regions. Among these kinases, Plk1 has the most remarkable impacts on BRCA2. Plk1 is involved in cell cycle progression and mitosis regulation and it is found...
Relaxation dispersion techniques are powerful tools to quantitatively characterize the chemical (or conformational) exchange across biologically relevant timescales [Palmer et al, Meth. Enzymol. 2001 & 2019]. Recently, a new type of data analysis, geometric approximation methodology [Chao & Byrd, JACS 2016], has been developed to decipher the complex experimental data associated with the...
We have recently shown that the hyperpolarized material obtained by dynamic nuclear polarization (DNP) at cryogenic temperatures can be transferred from the polarizer magnet to a target magnet in the solid form while maintaining a high level of the nuclear spin polarization - this method is named bullet-DNP [1]. This approach allows for rapid sample transfer and minimal dilution of the...
In Bullet-Dynamic Nuclear Polarization the sample is transferred from the polarizer to the target magnet as a solid. In the target magnet it is rapidly dissolved – transfer and dissolution take approximately $900\ \mathrm{ms}$. As we have previously reported we can achieve liquid-state $^{13}$C polarization levels of $30\%$ at less than 10-fold dilution while using sub-mL solvent volumes as...
UFM1 is an ubiquitin like protein (UBL), which shares high structural but little sequence similarities to ubiquitin. Like ubiquitin it can be covalently attached to target proteins by specific enzymatic cascade called ufmylation. Ufmylation of target proteins plays a role in central cellular events, such as gene transcription, response to cell stress and differentiation. The process of...
RIP2 is an intracellular adaptor protein that is essential for several reaction cascades. RIP2 is recruited when the bacterial fragments activate the innate immune pattern recognition receptors NOD2 and NOD1. That leads to NF-κB associated activation of proinflammatory cytokine production. Also, RIP2 involved in signal transduction by transmembrane neurotrophin receptor P75. That pathway is...
The high relevance of G protein-coupled receptors (GPCRs) becomes apparent from their diverse distribution in human tissue and their participation in a variety of physiological functions. This makes them highly interesting molecules for basic research and also a major target for pharmaceutical drugs.
Besides the structural architecture of the seven transmembrane proteins and its interaction...
Uropathogenic Escherichia coli invades and colonizes hosts by attaching to cells using adhesive pili on the bacterial surface. Although many biophysical techniques have been used to study the structure and mechanical properties of pili, many important details are still unknown. Here we use proton-detected solid-state NMR experiments to investigate solvent accessibility and structural...
To decorate biotechnologically prepared proteins with chemically synthesized polymers opens the route to hybrid molecules and materials with novel and unique properties.
The protein component may provide highly specific biochemical activity whereas the polymer part may improve long-term stability, modulate bioavailability and enable the integration of biological function into bulk...
RNA is a versatile biomolecule that plays an important role in many cellular processes e.g. translating the genetic information, gene regulation, and reaction catalysis in organisms. NMR spectroscopy can be used to obtain conclusive informations about the structure and dynamics of a system, as well as to investigate interactions of RNA with proteins, ions or ligands. However, long RNAs with...
Cholera is an acute, diarrheal illness caused by infection of the intestine by the bacterium Vibrio cholerae. An estimated 2.9 million cases and 95,000 deaths occur each year around the world. During infection, the bacterium releases a toxin. The cholera toxin is an oligomeric complex made up of six protein subunits: one copy of the enzymatic A subunit and five copies of the receptor binding B...
Channelrhodopsin-2 (ChR2) is a light-gated ion channel[1] which over the last years has attracted considerable interest due to its unparalleled role in optogenetic applications[2]. However, despite considerable efforts, it has remained elusive how molecular events during the photocycle including the retinal trans-cis isomerization and the de/re-protonation of the Schiff base are coupled to the...
Recently, the proposal and concept development of using chip-integrated voltage-controlled oscillator for electron spin detection has gained significant attention in the research community. Being at the same time highly sensitive and compact in size, these ESR-on-a-chip detectors are ideally suited for next generation portable ESR spectrometer [1]. Moreover, thanks to the fast electronic...
Gating modifier toxins (GMTs) from spider venoms may serve as pharmacological hits for treatment of hereditary diseases caused by mutations in genes coding voltage-gated sodium channels (Nav). Nav channels are transmembrane proteins, consisting of one pore domain and four surrounding voltage-sensing domains (VSDs). In particular, mutations in the S4 transmembrane helical segments of the VSDs...
Acylgermanes are efficient germanium-based photoinitiators (PIs) that are suitable for medical applications such as dental fillings.[1–4] Remarkably, this class of compounds displays electron-transfer properties being comparable with phosphorus-based photoinitiators.[5]
Here we present a combined EPR/DFT/CV approach to characterize the redox properties of acylgermanes....
In view of the world-wide emergence of pathogens being resistant to a large range of commonly used antibiotics finding pharmaceuticals with novel mechanisms of action has become an urgent need. Antimicrobial peptides are abundant in the plant and animal kingdom, have escaped resistance development over millions of years and are therefore considered valuable templates to develop novel drugs. In...
Nuclear magnetic resonance (NMR) spectroscopy is a very powerful tool of investigation in structural biology, providing structural and dynamic information at atomic resolution on a protein either alone or in its interaction with small molecules or with other proteins/nucleic acids [1-2].
Electron Paramagnetic Resonance (EPR) spectroscopy well complements NMR data providing information useful...
Intramembrane proteases catalyse the hydrolysis of their substrates within the hydrophobic environment of the lipid bilayer and are involved in several diseases and the functional activation of substrates [1]. SPPL proteases belong to the class of GxGD aspartyl proteases. It is still unknown how members of this family distinguish between substrates and non-substrates, or which factors...
The vast volume of sea oil transport and rise in the number of underwater drilling platforms all around a world creates a great risk for environment. To minimize the ecological impact of possible water spills there is a need for cheap, oil selective materials used for cleaning oil spills from the sea surface.
In the recent years a new branch of aerogel material made up of cellulose...
The bioactivities and physical-chemical properties of chitosan are strongly dependent on the contents of 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) and 2-amino-2-deoxy-D-glucopyranose (GlcN) units, which define the average degree of acetylation (DA). In this study, high molecular weight chitosans with variable DA grades (5 to 60%) were produced by multistep ultrasound-assisted deacetylation...
Neurotensin (NT) is a 13 amino acid peptide expressed in the central nervous, gastro-intestinal and cardiovascular systems where it acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate, primarily through activation of neurotensin receptor 1 (NTS1), a G Protein-Coupled Receptor (GPCR). Peptide ligands of GPCRs bind through complex, and possibly multiple modes...
Riboswitches are RNA structures that regulate the downstream gene expression upon binding of a small molecule ligand. The guanidine-II riboswitch is a guanidine-binding translational ON switch that is present in many proteobacterial species. The associated genes are usually involved in guanidine detoxification. This riboswitch class is characterized by two hairpins (P1 and P2) with an ACGR...
The Two-Partner Secretion (TPS) pathway in Gram-negative bacteria is dedicated to the export of large proteins serving notably as virulence factors. TpsB transporters are transmembrane β-barrel proteins secreting their TpsA substrates across the outer membrane. They belong to the ubiquitous Omp85 superfamily mediating protein insertion into or translocation across membranes. In the whooping...
G protein-coupled receptors (GPCRs) are membrane-integrated signaling proteins that translate extracellular stimuli into an intracellular response for processes ranging from vision, smell and taste to immunologic, neurologic and reproductive functions. Only 16% of the superfamily are clinically targeted – and yet these comprise nearly 35% of all marketed drugs. Despite their obvious...
Conformational ensembles of intrinsically disordered proteins (IDPs) are particularly challenging to characterize. One can obtain information about the entire conformational ensemble of the IDPs by Solid-State NMR in frozen solution. From the experimental side, we have studied the sparsely isotope labelled IDP a-synuclein (α-syn) in frozen solution by Dynamic Nuclear Polarization NMR (DNP-NMR)...
Aromatic side chains are attractive probes of protein dynamics on the millisecond timescale, because they are often key residues in enzyme active sites and protein binding sites. Furthermore, they allow to study specific processes, like histidine tautomerization and ring flips. Here we investigate the possibility of aromatic 1H CPMG relaxation dispersion experiments as a...
2D NMR spectroscopy is an extremely useful method for analyzing the structure and dynamics of small molecules in liquids and in solid state. Modern 2D NMR approaches allow obtaining direct information about the geometrical configuration of small molecules, which is very important when solving problems of modern physical chemistry of fluids (e.g. micronization of drug substances,...
Several human diseases result from malfunctions of ABC systems. Many ABC exporters contain asymmetric nucleotide-binding sites (NBSs) and some of them are inhibited by the transported substrate.[1] To actively transport diverse chemically substrates across biological membranes, ATP-binding cassette (ABC) transport complexes use the energy of ATP binding and subsequent hydrolysis. In this work,...
Conventional nuclear magnetic resonance (NMR) experiments are limited by low sensitivity. Hyperpolarization techniques such as dynamic nuclear polarization (DNP) enhance NMR signals by several orders of magnitude [Larsen et. al. 2003]. Most dissolution-DNP (dDNP) applications involve the use of weakly magnetic isotopes such as 13C, but excessively long...
Conjugated organic molecules exhibit unique optical, magnetic and electrochemical properties, and have been used for organic molecular magnets and organic thin-film devices such as solar cells, organic light emitting diodes (OLED), or organic field effect transistors. Their properties are strongly affected by the spin structures, which can be effectively investigated by Electron Spin Resonance...
Since the FDA approval of Amphotericin B (AmB) in 1959, this polyene antifungal drug has become the primary treatment for systemic fungal infections due to its broad-spectrum antifungal activity and evasion of antimicrobial resistance1. However, the mortality rate for immunocompromised patients with systemic fungal infections remains high due to AmB’s toxicity1....
The dangling bond (db) is the most prominent electronic defect in hydrogenated amorphous silicon (a-Si:H). Acting as recombination centers for charge carriers, dbs crucially determine the electronic material properties. Understanding the mechanism of db-related recombination thus constitutes one of the most fundamental issues concerning a-Si:H.
Since a neutral db is paramagnetic, the ideal...
Some quantum computation schemes rely on having ordered structures of qubits with nano-scale positioning accuracy to perform correctly. One natural candidate for such a system of qubits is an ordered structure of electron spins in a solid-state sample. Such systems (e.g. $\text{NV}^{-}$ centers in diamond, and phosphorus doped $^{28}\text{Si}$) exist in unordered forms, and have been shown to...
Delta subunit of RNA polymerase is exclusively present in Gram positive bacteria. It was proven that it is important for virulence of the bacteria, it ensures the quick and proper response to the changing cell environment conditions, and its role for regulation of RNA polymerase activity was also demonstrated [1]. However, the function of the delta subunit is not fully understood at molecular...
A wide ensemble of protein-protein interaction networks warrants communication and information exchange between cells. These interactions are mainly mediated by protein domains, which could be considered as protein modular building blocks assembled in different fashions. They decode specific signals emerging from post-translational modifications involved in receptor signaling, endocytosis or...
Depsi peptides are known to undergo an intramolecular O-N-acyl migration at physiological conditions triggering peptide nanofiber growth. Based on this process, our group has recently developed biocompatible, injectable, and self-healing hydrogels that are crosslinked by the coagulating peptide nanofibers. With these properties, together with the ability for a pH-controlled gelation, depsi...
Hyperpolarization by dissolution dynamic nuclear polarization [1] has emerged in the last decade as a powerful tool for boosting the sensitivity of magnetic resonance by orders of magnitude. This has for instance brought metabolic imaging to reality [2]. Unfortunately, hyperpolarization’s lifetimes in all molecules hardly exceed a minute. This means that hyperpolarization needs to be produced...
Heterogeneous catalysis offers intrinsic benefits for parahydrogen induced hyperpolarization (PHIP). In addition to the ease of separation of the catalyst and compatibility with continuous-flow production of hyperpolarized fluids, supported transition metal nanoparticle catalyst materials are generally more robust and recyclable than dissolved transition metal complexes. However, the down-side...
Paramagnetic restraints have been used in Biomolecular NMR for the last three decades in order to elucidate and refine structures but also to characterize protein/ligand interactions1. Pseudo Contact Shifts (PCS) are the most commonly used restraints, consisting in measuring a shift between a paramagnetic species and a diamagnetic reference, whose magnitude depends on the distance...
Recently the access to 19F-containing screening libraries has started a great pharmaceutical interest for 19F-based screening methods. Especially the 19F-CPMG experiment can be used for detection of protein-ligand interactions. However, when screening large libraries of fluorinated organic compounds, pulses that cover the 19F-bandwidths of 120 kHz on...
Microwave driven dynamic nuclear polarization (DNP) is one of the successful approaches to overcome the sensitivity limitations of NMR, opening new possibilities and applications in materials and life sciences. The design of new and ever more efficient polarization sources has been accompanying the successful development of high field DNP. However, the radical improvements are performed...
Site-directed spin labeling (SDSL) is a powerful technique to monitor highly dynamical conformational states of membrane proteins in both NMR and EPR. In this work, the nitroxide spin label MTSL was introduced to single cysteines in the G protein coupled neuropeptide Y2 receptor (Y2R) that has been associated with a number of key physiological functions, including the regulation of appetite...
The relationship of pancreatic cancer (PC) and diabetes mellitus (DM) is obvious, as more than 2/3rd of the cancer cases are diagnosed with DM. This so-called pancreatogenic diabetes (T3cDM) appears prior to PC within a period of three years and is accompanied by characteristic features such as absence of obesity or frequent infections1. PC belongs to the most lethal...
Intrinsically disordered proteins (IDPs) are enriched in “disorder-promoting” residues such as proline. In solution Xaa-Pro bonds can undergo a cis-trans isomerization. Functional studies show the biological relevance of the different types of conformation.1 The cis-isomer is less frequent (5-15 %) due to steric hindrances and effects also the proline surrounding residues....
Ferredoxins (Fdxs) are soluble iron-sulfur proteins that function as electron acceptors and donors in diverse metabolic pathways. They are characterized by their acidity and low redox potentials ( 230 to 420 mV). The unicellular green alga Chlamydomonas reinhardtii contains 12 different plant-type [2Fe2S]-Fdx isoforms.$^1$ The most abundant isoform Fdx1 has been characterized extensively...
We have demonstrated 57Fe Zero Field Nuclear Magnetic Resonance (ZFNMR) as a powerful tool in determining the structural composition of nickel-cadmium spinel ferrites of various compositions of Ni1-x CdxxFe2O4 from x = 0 to1, which are synthesized via one-step auto combustion technique. The XRD measurements confirm the phase purity of...
Solid-state dynamic nuclear polarization (DNP) NMR utilizes radical-bearing polarization agents to enhance the signal of nearby nuclei. Consequently, small sample sizes along with insensitive and higher-dimension experiments become feasible on realistic time scales. These sensitivity gains are particularly intriguing for applications to biological systems, where the molecule of interest is...
The pulsed dipolar EPR spectroscopy (PDS) techniques such as double electron-electron resonance (DEER) or relaxation-induced dipolar modulation enhancement (RIDME) detect the magnetic dipolar interaction within pairs of paramagnetic species from which the spin–spin distances and distance distributions can be extracted. By combination with site-directed spin labeling (SDSL) PDS became a...
The electrolytic reduction of carbon dioxide (CO2) is a promising option for the future of sustainable energy as chemical energy storage. Specifically, the electrolysis of CO2 in aqueous media using silver electrodes yields carbon monoxide (CO), which can be used to produce CO2-neutral fuels via Fischer-Tropsch synthesis. The technical implementation of this...
Heteronuclear-detected NMR experiments are becoming increasingly more common due to the constant development in cryogenic probes. These experiments are particularly beneficial with respect to nucleic acids due to the inherent low proton-density and limited 1H-chemical shift dispersion. Here, we present 15N- and 13C-detected NMR experiments that enable...
We have been developing a millimeter (mm)-wave ESR/NMR double magnetic resonance system in ultra-low temperature and high magnetic field regions aiming for direct detection of diluted nuclear spins.
Increase of nuclear magnetization by using dynamic nuclear polarization (DNP) may enable direct observation of NMR.
One of our targets is a silicon crystal doped lightly with phosphorus...
Fast field cycling (FFC) NMR relaxometry enhanced by DNP [1] has recently been reported to obtain T1 relaxation time dispersion with increased sensitivity and selectivity to study molecular dynamics in various systems. First studies were devoted to the measurement of the DNP spectrum and enhancement as well as DNP-enhanced dispersion curves of polymer systems in the presence of...
Cyclosporin A, well known as an immunosuppressive agent, is one of a family of congeners synthesised in certain soil fungi. Three natural variants of cyclosporin (B, C, D) were studied by high resolution NMR spectroscopy in chloroform and dimethyl formamide. CsB has alanine in position 2 instead of $\alpha$-aminobutyric acid (Abu2) found in CsA, in CsC this residue is replaced by threonine,...
Type II collagen is the most abundant macromolecule in articular cartilage. In the tissue collagen forms anisotropic, fibrillous structures. Longitudinal relaxation is generally thought to be isotropic in articular cartilage [1]. We investigate the conditions needed to observe the anisotropy of the collagen network in the longitudinal relaxation time $T_1$.
We have computed longitudinal...
With a staggering energy density of 142 MJ kg$^{-1}$, three times larger than that of petroleum, hydrogen became one of the most valuable and environmentally-friendly fuels [1]. Hydrogen is an energy carrier that does not suffer from energy dissipation (electricity as a direct energy source does), since the energy is chemically stored; however, it lacks of adequate means of confinement and...
Within the last 15 years dynamic nuclear polarization (DNP) has emerged as an efficient tool to boost the sensitivity of various magnetic resonance techniques[1]. Dissolution DNP has proven its applicability in magnetic resonance imaging (MRI) as well as solid-state DNP in nuclear magnetic resonance (NMR)[1]. However, liquid state NMR DNP is still in its infancy. Even though the general...
We apply solid-state MAS NMR-spectroscopy at different temperatures to investigate protein folding and misfolding on a molecular level [1].
On the one hand, we exploit DNP-enhanced MAS NMR spectroscopy at low temperatures (~100K) to investigate conformational ensembles of intrinsically disordered proteins IDPs). Such proteins are not represented by a single well-defined structure but rather by...
The ability of corals to maintain homeostasis and mineralize has been compromised by ocean acidification and temperature rise causing reefs to recede and even vanish. Therefore, corals have been serving lately as important proxies of environmental impact on marine life. As newborn, corals undergo transformation from a swimming organism (planula) to an benthic immobile one (polyp), that lives...
Enhancement of NMR signals using dynamic nuclear polarization (DNP) enables studying systems that are difficult to tackle with conventional NMR due to low concentrations or low overall quantities of material. One particular example is amyloid-beta peptide, which self-assembly in human brain tissue leads to neurodegeneration in Alzheimer’s disease (AD). Previously, various intermediates along...
Proteorhodopsin (PR) is found in marine bacteria in various ecosystems and is one of the most abundant photoreceptors. It converts light into a transmembrane, electrochemical gradient and forms functionally unresolved higher oligomers. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific...
Accurate geometrical tracking of the tip of a catheter during minimal invasive surgery using intervention MRI provides a surgeon with orientation relative to the body. The tip can be visualized in the MR environment by locating a micro-resonator at this position. If the resonator is tuned to the Larmor frequency of abundant nuclei, and subject to a radio frequency excitation pulse, it's motion...
Dynamic nuclear polarization (DNP) has proved to be a powerful and versatile means to overcome the intrinsically low sensitivity of NMR[1]. A great challenge for DNP at present is to achieve high spin polarization in shorter times, which requires understanding spin polarization transfers but also spin diffusion dynamics[2].
Spin diffusion is indeed of major importance nearby the free...
β-lactamases pose a great threat to our healthcare system, yet may simultaneously present potential solutions. We used CEST, CPMG relaxation dispersion and T1, T2, NOE Lipari-Szabo analyses to characterise the dynamic behaviour of the β-lactamase of Mycobacterium tuberculosis, BlaC, over a wide range of timescales. BlaC is mostly rigid on the fast timescales and shows...
We investigate the dynamics of CO2 adsorbed in a metal-organic framework (MOF) in high pressure range (~1 MPa) utilizing the combination of MAS NMR and CSA analyses. MAS NMR spectra show adsorbed CO2 adsorbed and free mobile gas-phase CO2. Analyses for the dynamics by T1 measurement suggest that the adsorbed CO2 in pores has very slow mobility corresponding to the values reported for some...
The activities of membrane proteins strongly depend on their dynamics; herein we made use of proton-detected MAS at very high-field and of relaxation violated experiments in liquid state NMR to describe the dynamics of the membrane domain of the Outer Membrane Protein A of Klebsiella pneumoniae (KpOmpA) in liposomes and in micelles.
From 15N relaxation rates and...
The 117-residue GABAA receptor-associated protein (GABARAP) from H. sapiens is a versatile key regulator in autophagy. GABARAP is found as a soluble protein in the cytosol, as well as anchored to autophagosomal membranes during autophagosome formation and maturation. Anchoring to phospholipid membranes is achieved via enzymatic C-terminal lipid-conjugation of GABARAP, but the...
We consider the behaviour of precessional phase carried by molecules of a diffusing specimen under magnetic fields typical of magnetic resonance experiments. A time evolution equation for the ensemble of particles is constructed, which treats the phase as well as the position of the molecules as random variables. We refer to the associated probability density function $p ({\vec x}, \phi |t)$...
Oncogenic variants of kras gene are among the most important mutations causing cancer. KRas protein is a membrane-bound small GTPase which acts as a molecular switch and plays a key role in many signal transduction pathways regulating cell proliferation, differentiation, and survival. It alternates between its GTP-bound active and the GDP-bound inactive conformers. The most frequent oncogenic...
Polyethylene terephthalate (PET) is one of the most widely used plastics worldwide and is applied, i.a., as packaging material in the food industry.[1] Like many polymers, PET is susceptible to photochemical degradation and absorbs in the ultraviolet (UV) range.[2]
As already known, UV irradiation of PET leads to shorter molecular chains and thus to a higher number of...
Purpose: 4D flow magnetic resonance imaging (MRI) allows to measure and visualize blood flow in vivo . This is in particular interesting for the investigation of the effect of flow diverter stents used for treating aneurysms. However, metallic stents induce artifacts on MRI like other metal objects. It was realized before that the effect of these artifacts on the measured flow is important but...
Dynamic Nuclear Polarization (DNP) is the most widespread and ubiquitous technique for signal enhancement in Nuclear Magnetic Resonance (NMR). Introduction of the ability to arbitrary modulate the 193 GHz waveform used for excitation of the electron spins at 7 Tesla resulted in up to a factor of 4 improvement in DNP efficiency under varying range of experimental conditions and across different...
G-quadruplexes are structurally diverse non-canonical secondary structures formed by guanine-rich nucleic acids capable of performing a broad range of cellular functions, most notably regulation of gene expression. As independent aptamer molecules, G-quadruplexes can also bind specific cytosol or extracellular proteins, which makes them promising drug targets for treatment of various diseases....
So far, we investigated the electronic structure of novel type of organic conductors, ammonium tetrathiafulvalene carboxylate (TTFCOO) and its and tetrathiapentalene derivative (TTPCOO) by high-field ESR and NMR measurements. The pristine TTFCOOH and TTPCOOH molecules are closed-shell. Kobayashi and coworkers (NIMS, Japan) found that self-doped type carrier was generated by substitution of the...
Complex molecular mixtures of biological origin are difficult to analyze. Arabinoxylan oligosaccharides (AXOS), cereal derived dietary fibers, are a typical example. 1H-DOSY-NMR is frequently used for determining mixture composition by diffusional separation, but in the case of AXOS, it falls short because of severe overlap of signals due to extensive presence of J-couplings, and...
Collagens are the most abundant components of the extracellular matrix (ECM). Due to their diverse structures and compositions, collagens serve many functions, providing structural and mechanical support for surrounding cells, and playing important roles in cell-to-cell communication. Nonetheless, despite being at first glance a simple protein formed by three homologous polypeptide chains of...
We have demonstrated that 1H spin–spin relaxation time (T2), which is measured by using low field NMR techniques, is an effective parameter to elucidate the degradation behavior of poly (urea-urethane). It is difficult to reveal the degradation behavior of poly (urea-urethane), when the degradation proceeds in urea or urethane bond units.
We prepared two types of poly...
Pyrrolo[1,2-a]benzimidazole and pyrrolo[1,2-a]quinoxaline skeletons are present in various bioactive compounds. Thus, pyrrolo[1,2-a]benzimidazole scaffold is present in a range of DNA cross-linkers mimicking the mitomycin antitumor activity against a range of human cell lines, and many pyrrolo[1,2-a]benzimidazole derivatives showed beneficial properties on central nervous system...
β-peptides are important peptidomimetic compounds. Here the formation of ordered backbone conformation of new pentamer β-peptides was studied by NMR (1H, ROESY, TOCSY, COSY NMR techniques) and ECD spectroscopy. The investigated pentamers include trans-[R,R]-2-aminocyclohexanecarboxylic acid (trans-[R,R]-ACHC) building blocks interrupted by different elements in the middle of...
Currently, proton nuclear magnetic resonance (1H-NMR) have been combined with electrochemistry (EC) to observe reaction pathways and identify products generated due to its high selectivity, capacity in chemical structure determination [1], and quantitation [2]. We have utilized NMR in our electro-catalysis researches, with a representative example that electro-catalytic performances of...
Nuclear magnetic resonance (NMR) spectroscopy serves as a versatile analytical tool. Resolution is an essential challenge in NMR spectroscopy. Due to the limited range of chemical shifts and extensive signal splittings due to scalar couplings, signal congestion and even overlap are common in NMR spectra, especially in the widely used proton spectra. One major approach to enhance spectral...
The present reports the results of the experimental study of static and dynamical characteristics of the Er3+ ions in the YGa3(BO3)4 crystals. g-factors and the constants of hyperfine interaction are found. The ratio gzAx/ gx*Az is close to unity that is an evidence of insignificant admixing of the excited multiplets. The widening of the absorption lines related to the temperature increase...
Amino acid radicals are involved in essential processes in primary metabolism such as photosynthesis, respiration, and biosynthesis of DNA building blocks. They serve as one-electron redox cofactors in biocatalysis and multistep proton-coupled electron transfer (PCET) reactions. E. coli class Ia ribonucleotide reductase (RNR), which catalyzes the reduction of nucleotides to their...
Hsp90 is a central chaperone performing its activity hydrolyzing ATP with Mg(II) as cofactor. Hsp90 is homo-dimeric and each monomer consists of 3 domains (CTD, MD, NTD). The ATPase site is in the NTDs, while the CTDs are dimerized. In absence of nucleotide Hsp90 is in ‘open’ conformation shifting to an ATP-bound ‘closed’ conformation by dimerization of the NTDs.[1] Yet, there are still open...
Proteins are increasingly being used in numerous technological applications such as biosensors, biofuel cells and biocatalysts. Often the immobilization of a protein in a synthetic matrix is essential.1,2 Porous matrices have warranted much attention due numerous factors, such as their high surface areas, meaning improved protein loading and high number of active sites; the...
Dense metal-formate frameworks of general formula [A][M(HCOO)3]n attracted significant attention of the scientific community due to the indications of the multiferroic behavior [1]. These compounds consist of transition metal ions (M2+) linked by formate groups into porous frameworks, where each pore confines a single molecular cation (An+). The...
Riboswitches are non-coding RNAs binding a specific second-messenger, modulating transcriptional or translational efficiency of their corresponding genes. These regulatory mechanisms are found in a wide variety of bacteria and are often associated with lifestyle changes like biofilm formation and transition into anaerobic metabolism.[1-3] These lifestyle changes include pathways required for...
Pyrimidine base namely Uracil and its derivatives have immense biological relevance. The Proton Transfer (PT) rate in solution for these molecules determines the nature of solvation, cellular mobility and effect of solution pH on the biological activity. Therefore, quantification of PT rate has attracted attention over the years[1,2]. In the present study, an attempt has been made to avail...
RNA does not only translate the genetic code into proteins, but also carries out important cellular functions. Understanding such functions requires knowledge of the structure and dynamics at atomic resolution. Almost half of the published RNA structures have been solved by NMR, but as a result of severe resonance overlap and low proton density, high-resolution RNA structures are rarely...
The rapid methodology development of sensitivity-enhanced SSNMR spectroscopy has been empowering its success in a broad range of challenging fields in life science. Here I will present a new methyl SSNMR toolkit for exploring the protein structures that offers new types of NMR parameters for decoding the complex molecular systems. These approaches meld DNP-enhancement, heteronuclear NOE, tamed...
A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular Dynamics (MD) has often been combined with force spectroscopy experiments to...
Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC) is a 24-amino acid residue host-defence peptide, first isolated from the skin secretions of the foothill yellow-legged frog Rana boylii. The peptide is of interest as it shows broad-spectrum antimicrobial activity, and is particularly effective against opportunistic yeast pathogens. Its potential for clinical use, however, is hindered by its latent...
Arkadia is a RING E3 ubiquitin ligase that positively regulates TGF-β signaling pathway by mediating degradation of the negative regulators Smad6 and Smad7 and the nuclear co-repressors Ski and Skil (SnoN)1,2. The domains that are required for the substrate recognition and ubiquitin ligase activity are located in the highly conserved area of the C-terminal 100 amino acids. This region is...
We propose an easy and precise method of quantitation by NMR, using the neat protonated solvent as external concentration reference. Spectra of the analyte in a deuterated solvent, e.g. chloroform-d, and the neat solvent, in this case protonated chloroform, are taken in the same conditions, i.e. the same relaxation delay, pulse width, acquisition time and receiver gain. The linearity of the...
Smilacis Glabrae Rhizoma is a traditional Chinese medicine used for dissipating stasis, detoxification and diuresis. Astilbin and its three diastereoisomers (neoastilbin, isoastilbin, and neoisoastilbin) and engeletin are the main bioactive components. To quickly conduct the quality control, a proton nuclear magnetic resonance approach was established to simultaneously quantify the absolute...
Because of the hyperfine coupling between unpaired electrons and nuclear spins, NMR signals in paramagnetic systems often experience large shifts and possess short relaxation times, particularly in close proximity to the metal center, rendering observation of active sites in metalloproteins particularly challenging.
Here, we illustrate how, by adapting a set of NMR experiments originally...
Protein-water interactions have widespread effects on protein structure and dynamics. As such, the function of many bio-macromolecules can be directly related to the presence and exchange of water molecules. While the presence of structural water molecules can be easily detected by X-ray crystallography, the dynamics within functional water-protein networks is largely elusive. Here we use...
We investigate the feasibility of performing functional MRI (fMRI) at ultralow field (ULF) with a Superconducting QUantum Interference Device (SQUID), as used for detecting magnetoencephalography (MEG) signals from the human head. While there is negligible magnetic susceptibility variation to produce blood oxygenation level-dependent (BOLD) contrast at ULF, changes in cerebral blood volume...
NMR spectroscopy has proven a valuable tool in differentiation of the geographical origin of various food products.[1] The geographical origin is influenced by the composition of metabolites due to different exogenous factors such as soil, temperature or water availability. The concept of a metabolomics-based approach based on the determination of a molecular fingerprint which is...
NMR spectroscopy provides detailed insight into molecular structures and is capable of quantitation without standards. An area of interest for chemical industry is the possibility to use NMR to rapidly analyze wastewater streams with anomalously high TOC (Total Organic Carbon). A high TOC excursion can lead to regulatory violations and the consequent danger of shutting down an entire...
A fragment screen using ligand detected 19F NMR versus a kinase target will be described. In addition, practicalities of sample preparation and mixture design will be discussed. Furthermore, a TopSpin/Perl script package for automatic analysis of 19F NMR based screening data will be presented.
Among the translated proteins are few validated drug targets. A recent estimate, based on currently approved drug substances, listed 218 validated protein targets. The choice of non-coding RNA as a drug target could be an alternative approach to efficiently control biomolecular signaling pathways. Currently, clinical trials are underway with oligonucleotides and small molecule drug candidates...
Iron(III) porphyrins play vital roles as catalysts, protein cofactors, and single-molecule magnets (SMMs). SMMs represent potential candidates for spin-based nanoscopic data storage due to slow relaxation of the magnetization below their blocking temperature. Since the zero-field splitting (ZFS) parameters D and E strongly affect the properties of intermediate- and high-spin ferric...
Toxin-antitoxin (TA) systems are essential for bacterial persistence under stressful conditions. In particular, M. tuberculosis express VapBC TA genes that encode the stable VapC toxin and the labile VapB antitoxin. Under normal conditions, these proteins interact to form a non-toxic TA complex, but the toxin is activated by release from the antitoxin in response to unfavorable conditions....
Guanine-rich DNA oligonucleotides can adopt non-canonical, four-stranded secondary structures, termed G-quadruplexes. The formation of G-quadruplexes requires the presence of cations. In fact, cation nature is one of the major factors contributing to the structural diversity of G-quadruplexes. Their self-assembling ability, electrochemical properties and programmable control of their shape and...
The undisputed usefulness in biomedical applications, the high carbon polarization (up to 70%) and the long relaxation time constant after dissolution have made neat [1-13C]pyruvic acid (PA) plus trityl the most studied dissolution DNP sample.
Therefore, sometimes the literature is lacking detailed studies for different trityl-based samples, limiting the potential of other...
Fifteen years after its invention, dissolution dynamic nuclear polarization (dDNP)[1] has become one method of choice to enhance signals in both MRI and liquid state NMR. Despite exciting prospects, this method remains restricted to few research groups as it needs in principle to be performed at the point of use, with specialized equipment and personnel. However, a series of recent advances...
Hyperpolarization via dissolution DNP (dDNP) is a highly promising technique that has advanced into a powerful modality for non-invasive real time monitoring of normal and abnormal cellular physiology with the potential to unravel diseases, develop novel treatment regimes, and quantify enzymatic processes. 1, 2 An important enzyme in this context is branched chain amino acid...
Rapid-scan (RS) electron-spin resonance (ESR) is a technique to boost the signal-to-noise ratio (SNR) in ESR experiments by allowing the use of larger B1 magnitudes without saturating the sample. State-of-the-art rapid-scan setups [1] use fast field sweeps produced by modulation coils in combination with relatively low-Q resonators to achieve a large detection bandwidth. Due to effects of the...
Understanding the physico-chemical basics of protein-membrane interactions are of key importance in functional characterization of the cell membrane. In order to simplify this complex system the best membrane mimetics are bicelles, which at q=0.5 (long chain/short chain lipid) ratio are ideal for solution state NMR studies1,2.
We chose biologically relevant protein fragments: the...
Ionic liquids (ILs) and Deep Eutectic Solvents (DESs) are two strongly related classes of materials of great interest in green chemistry and with enormous potential in several technological and industrial applications.
ILs are well-known low melting salts with unique physicochemical features. To tune properties and structural organization of the final materials, there is nowadays a growing...
Availability of new materials, precision machining and good design software have facilitated the progress also in NMR probes, a key to successful experiment. Particularly interesting is a possibility to record 1H-MAS spectra at >170 kHz providing a 0.05 ppm homogeneous resolution which may become an alternative to, even supersede, the present “high- resolution” NMR. Despite a very small, ca...
South Africa’s coastline, stretching more than 2500 km from the cold temperate coast west coast to the warm tropical environment of northern Kwazulu-Natal, has an exceptional environment with a high percentage of endemic species. SA’s rich marine biodiversity therefore signifies the production of potent, biologically active, natural products (Davies-Coleman & Beukes, 2004). By 1995, 60% of the...
The Heme group is synthetized in a biosynthetic pathway by 8 enzymes. Defects on these enzymes lead to a family of diseases called Porphyria. To understand the consequences of the alteration of this route, it is important to gain knowledge about its regulation and the changes in its metabolites. The development of a mathematical model of the route will provide us the capacity of predicting the...
Dynamic Nuclear Polarization (DNP) is an emerging magnetic resonance technique to enhance the sensitivity of MAS NMR. In a typical MAS DNP experiment, several mechanisms are involved simultaneously when transferring the relatively large polarization of paramagnetic electrons to NMR active nuclei of interest. Recently SCREAM-DNP (Specific Cross Relaxation Enhancement by Active Motions under...
Monoclonal antibodies (mAbs) are among modern bio-therapeutics which are often needed and applied in high concentrations (>100mg/ml), especially for subcutaneous injections. EPR spectroscopy is among the few methods which can be applied directly at such conditions, without making concentration approximations. To the best of our knowledge for the first time, EPR spectroscopy is used to study...
Techniques of high-frequency electron paramagnetic resonance (EPR) and electron spin echo (ESE) were applied for investigation of non-Kramers Tb3+ centers in single crystals of yttrium aluminum garnet, Y3Al5O12 (YAG). The EPR-ESE techniques were created on the base of highly stable microwave bridges, quasiresonant system for supplying microwave power...
Huntigton’s disease (HD) is an incurable neurological disorder triggered by the aberrant expansion of the polyglutamine tract present in the huntingtin protein (HTT). The pathological version of HTT (with more than 35 consecutive glutamines) is prone to aggregate and form insoluble fibrils within neurons, affecting their normal functions. However, the precise mechanism by which the expanded...
The alarming rise of drug-resistant bacteria urgently calls for novel antibiotics. A particularly interesting class of antibiotics target essential Lipid II molecules that are present in bacterial membranes, killing even the most refractory bacteria at nanomolecular concentrations. Unfortunately, the native binding modes of these membrane-active antibiotics are unknown due to the enormous...
Among the various alternative DNA structures, G-quadruplexes exhibit a remarkable variety of topologies. In this study, two adjacent guanosine residues adopting syn glycosidic bond conformations in a G-quadruplex of (3+1)-hybrid type were substituted by anti-favoring 2’-F-riboguanosine nucleosides (FrG). Residue-specific 15N labeling along with 2D NOE,...
Geological disposal of high-level radioactive waste usually includes bentonite buffer materials. However, the long-term performance of the bentonites for the containment of the nuclear waste is being increasingly questioned due to specific issues associated with “homogenisation” occurring in these materials. Homogenisation is driven by erosion, variations in density and development of swelling...
Lithium is a leading drug for the treatment of bipolar disorder and affects many biochemical pathways via inhibition of phosphatases and kinases. Lithium can replace magnesium cations in enzymes and small molecules, among them ATP. Yet, despite its fundamental pharmaceutical importance, the mode of binding of lithium to ATP has never been directly observed. Here we present the binding...
Hyperpolarization techniques have the potential to improve the sensitivity of magnetic resonance imaging (MRI) by more than four orders of magnitude.1 Various hyperpolarization techniques have been used to generate hyperpolarized contrast agents to study and diagnose diseases in vivo.2 Amongst the challenges of designing hyperpolarized contrast agents, two are persistent: the need to create...
Hyperpolarized 129 Xenon NMR spectroscopy provides a powerful method to investigate porous materials as has been shown on silica-based materials of MCM-41 structure in previous work in our group [1]. It offers insights to pore sizes, pore geometries and diffusivity. We are planning to apply hyperpolarized Xenon NMR for probing surface-functionalized porous materials based on...
Magnetic Resonance Imaging (MRI) was proven to be a powerful tool for studying catalytic reactions in operando due to its non-invasive nature and versatility. However, MRI application for gas-phase reactions studies is particularly challenging because of low spin density of reactants and products, and it’s further hampered by inhomogeneity of the magnetic field, associated with the presence...
It has been demonstrated that hyperpolarization using PHIP can be effectively achieved using a ruthenium-based trans-hydrogenation catalyst in contrast to typical cis-hydrogenation reaction [1,2]. This led to a development towards hyperpolarized metabolites such as fumarate, using pairwise trans-hydrogenation of an unsaturated precursor [3]. Aside of the desired reaction to form...
Hyperpolarization techniques are used to enhance NMR signals by several orders of magnitudes. Para-Hydrogen Induced Polarization (PHIP) is a practical and cost-effective method with great potential for medical applications. Moreover, the hyperpolarization of biocompatible solvents has potential for versatile applications in structural biology and medical imaging. Our group recently...
Hyperpolarisation provides the means to dramatically increase the signal-to-noise ratio in an NMR spectrum, however, the useful lifetime of the hyperpolarized state is limited by relaxation processes that lead to the re-establishment of thermal equilibrium over time. In order to make hyperpolarisation useful for experiments requiring lengthy delays for magnetisation transfer, chemical exchange...
Studying intrinsically disordered proteins (IDPs) under physiological conditions may present a great challenge using methods based on HN detection. The main drawback is the signal loss caused by chemical exchange of amide protons with water. Thus, conventional triple-resonance methods such as HNCA, HNCACB and HNCO fail to provide satisfactory information. The alternative way of...
With the recent record power conversion efficiency of 17.3% [1], organic solar cells have become serious contenders among third generation photovoltaics. This success is related amongst other things to efforts in overcoming the drawbacks of organic semiconductors, such as their low exciton-dissociation yield and charge-carrier mobility, as they limit device performance.
Studying...
Proteins of the MAP1 (microtubule-associated protein) family are implicated in the regulation of microtubule spatial organisation and stability. One of its members is MAP1B, a predominantly neuronal protein required for neuronal network formation and synaptic maturation in murine brain development. [1],[2] Its importance is further highlighted by its implication in several human...
Abelson tyrosine kinase (Abl) is an ubiquitously expressed kinase associated with a number of cellular signaling processes. The regulatory core of Abl consists sequentially of the N-cap, the SH3 and SH2 domains, and the kinase domain (KD) N- and C-lobes. The N-Cap differs between two splice variants, 1a and 1b. Abl 1b is 19 residues longer and N-terminally myristoylated. Under physiological...
In the food industry, low-field NMR has become the standard for solid fat content (SFC) determination of edible fats. The T2-relaxation behaviour of a semi-solid sample distinguishes a fast and slow relaxing part after a 90°-pulse, hence an FID experiment, related to the solid fat and liquid oil, respectively. Yet, data acquisition only starts after the 90°-pulse and the dead time....
DEER on orthogonally spin labeled proteins [1] is a versatile technique that allows to retrieve a much greater information content from a single sample than DEER on proteins spin labeled with just single type of label. The reason behind is that orthogonal spin labels can be addressed independently in DEER experiments due to spectroscopically non-overlapping central transitions and/or distinct...
In recent years Dynamic Nuclear Polarization (DNP), a hyperpolarization technique that boosts the NMR sensitivity, has become a powerful tool for structural studies. DNP applications span from the solid-state NMR studies of proteins, to the development of heterogeneous catalysts. However, its use has been mainly limited to low magnetic fields, due to the unfavourable field dependence of the...
Yersinia Adhesin A (YadA) is a protein found in the membrane of Yersinia Enterocolitica, which is involved in a number of food-borne diseases including enterocolitis, acute enteritis, diarrhea, and mesenteric limphadentisis.
YadA plays an important role in the ability of Y. enterocolitica to colonize a host, by aiding in the autotransport of a head domain to the cell surface that can stick to...
Most of the structural and functional information about biological macromolecules is obtained from experiments in vitro, far from their physiological context. Elucidating the structure, dynamics, function and molecular interactions of such complex molecules in their cellular context is necessary to understand physiological processes. NMR spectroscopy is an upcoming technique to investigate...
DNA - the most important intercellular target for UV radiation - can be damaged through the direct absorption by nucleobases, or through photosensitized reactions with endogenous cellular chromophores. Guanine that has the lowest oxidation potential among all DNA components is the main target of one-electron oxidation reactions. Guanine radicals are supposed to be involved in the formation of...