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Protein Dynamics by Solid-State NMR with Ultra-Fast Magic-Angle Spinning: from Microcrystals to Amyloid Fibrils and Membrane Proteins

mar 10 juil 2018



Institut des Sciences Analytiques - Amphi CRMN


Soutenance de thèse de M. Tanguy LE MARCHAND du CRMN - Institut des Sciences Analytiques, sous la direction de M. GUIDO PINTACUDA

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Description générale

Despite impressive progress in solid-state NMR with magic angle spinning (MAS) for the determination of structure and dynamics of proteins, MAS NMR studies are still far from routine. My work therefore aimed at extending the possibilities of MAS NMR for applications on complex biomolecular. For this, I exploited the unique possibilities provided by high magnetic fields in combination with the newest MAS probes. These experimental conditions allow to boost the sensitivity of MAS NMR through 1H detection at high resolution and to enrich the palette of probes for protein dynamics.

The first part of the thesis reports on my contribution to the development of new strategies for backbone resonance assignment, for structure elucidation, and for investigation of backbone and side-chain dynamics. These methodologies significantly reduce the requirements in terms of experimental time, sample quantities and isotopic labeling, and enlarge the molecular size of systems amenable to NMR analysis.

The second part describes the application of 1H detected MAS NMR to evaluate the role of protein dynamics in problems such as amyloid fibril formation and transport of molecules across lipid membranes. I first addressed the amyloid fibril formation propensity of human β2-microglobulin. I performed comparative studies of backbone dynamics of the wild type protein as well as a D76N mutant in crystals, and determined some of the structural features of the fibrillar form. This allowed to identify the presence of pathological folding intermediates and to formulate hypotheses on the mechanism of fibrils formation. Finally, I studied the mechanism of action of the alkane transporter AlkL in lipid bilayers. The measurement of dynamics parameters in presence or in absence of a substrate highlights possible routes for molecular uptake and lays the basis for a more detailed mechanistic understanding of the process.

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