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Theses

Magic-angle Spinning NMR of paramagnetic metalloproteins

Abstract : Most of our understanding of metalloproteins derives from atomic or molecular structures obtained from diffraction methods on single crystal samples. However, not all proteins are amenable for diffraction studies, and even when a highly-resolved structure is available, often the nature of the metal ion, its coordination geometry or its oxidation state are not determined. The aim of the present thesis is the investigation of structural properties of metal sites in paramagnetic metalloproteins by Magic-Angle Spinning Nuclear Magnetic Resonance (MAS NMR). MAS NMR is a powerful technique for the investigation of biological systems, and may represent a direct probe of the structure at the active site of paramagnetic metalloproteins. However, it suffers from limited sensitivity and resolution when applied to nuclei close to a paramagnetic center.In this thesis, we address these limitations by developing NMR methods based on ultra-fast (60-111 kHz) MAS rates. A “toolkit” of suitably designed pulse sequences is built for the detection and the assignment of nuclei in close proximity of a paramagnetic center. State-of-the-art computational techniques are also employed to convert the experimental data into structural restraints for obtaining atomic-resolution geometries of active sites. We benchmark this approach with the study of Fe, Cu and Co sites in two microcrystalline proteins, and we also provide preliminary data on a non-diffracting divalent metal ion transporter in lipid membranes. We anticipate that the techniques described here are an essential tool to elucidate many currently unanswered questions about structure and function of metal sites in structural biology.
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https://tel.archives-ouvertes.fr/tel-02092006
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Submitted on : Tuesday, April 9, 2019 - 2:05:07 PM
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  • HAL Id : tel-02092006, version 2

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Andrea Bertarello. Magic-angle Spinning NMR of paramagnetic metalloproteins. Other. Université de Lyon, 2018. English. ⟨NNT : 2018LYSEN004⟩. ⟨tel-02092006v2⟩

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