25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

Protein resonance assignment without spectral analysis: five-dimensional spectroscopy of immobilized proteins at ultrafast MAS

29 Aug 2019, 11:05
25m
Lecture Hall D (Henry Ford Building)

Lecture Hall D

Henry Ford Building

Talk Solid-state NMR development and applications Solid-state NMR Methods

Speaker

Dr Jan Stanek (Biological and Chemical Research Centre, Faculty of Chemistry, University of Warsaw)

Description

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

Primary authors

Dr Jan Stanek (Biological and Chemical Research Centre, Faculty of Chemistry, University of Warsaw) Mr Henry W. Orton (Research School of Chemistry, Australian National University) Dr Tobias Schubeis (Centre de Résonance Magnétique Nucléaire à Très Hauts Champs (FRE 2034 CNRS, UCBL, ENS Lyon), Université de Lyon) Mr Dylan Foucaudeau (Centre de Résonance Magnétique Nucléaire à Très Hauts Champs (FRE 2034 CNRS, UCBL, ENS Lyon), Université de Lyon) Mrs Claire Ollier (Centre de Résonance Magnétique Nucléaire à Très Hauts Champs (FRE 2034 CNRS, UCBL, ENS Lyon), Université de Lyon) Dr Tanguy Le Marchand (Centre de Résonance Magnétique Nucléaire à Très Hauts Champs (FRE 2034 CNRS, UCBL, ENS Lyon), Université de Lyon) Dr Rafal Augustyniak (Biological and Chemical Research Centre, Faculty of Chemistry, University of Warsaw) Dr Diane Cala-De Paepe (Centre de Résonance Magnétique Nucléaire à Très Hauts Champs (FRE 2034 CNRS, UCBL, ENS Lyon), Université de Lyon) Prof. Isabella C. Felli (CERM and Department of Chemistry, University of Florence) Prof. Roberta Pierattelli (CERM and Department of Chemistry, University of Florence) Prof. Sebastian Hiller (Biozentrum, University of Basel) Dr Wolfgang Bermel (Bruker BioSpin GmbH) Dr Guido Pintacuda (Centre de Résonance Magnétique Nucléaire à Très Hauts Champs (FRE 2034 CNRS, UCBL, ENS Lyon), Université de Lyon)

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