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Développement et applications de protocoles de synthèse in vitro du canal mécanosensible bactérien à large conductance, pour son étude structurale par résonance magnétique nucléaire en phase solide

Abstract : Membrane proteins account for almost 30% of the proteome and play essential roles in many cellular mechanisms. The mechanosensitive channel of large conductance MscL is an intrinsic membrane protein of the inner membrane of Escherichia coli. This protein acts as a valve pressure, in a hypo-osmotic down-shock, to prevent the bacterial lysis. In this work, a study of the structural characteristics of MscL in its native environment, the lipid bilayer, by nuclear magnetic resonance in the solid-state is presented. A major problem in determining the three-dimensional structure of membrane proteins by this technique is their overexpression and the analysis of carbon-carbon correlations from uniformly labelled samples. The analysis of MscL by solid-state NMR with a uniform labelling showed spectra with a good resolution, but the overlap of the correlations prevented any identification or assignment of the residues. We showed that selective labelling of the membrane protein MscL by using cell-free synthesis represents a crucial help in improving data sets obtained by NMR in the solid-state. Since the amino acids chemical shifts show a big overlap, different strategies to choose specific labelling patterns to get well-resolved spectra were developed. These strategies are based on chemical shifts prediction, or sequence analysis to isolate unique amino acid pairs. A combined approach was tested. These different approaches showed spectra with a good resolution, and facilitated the identification of amino acids in some cases. The methods developed in this work may gradually help to determine the three-dimensional structure of the mechanosensitive channel MscL, as well as other lipid-dependent membrane proteins.
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https://tel.archives-ouvertes.fr/tel-00621192
Contributor : Alaa Abdine <>
Submitted on : Friday, September 9, 2011 - 3:46:58 PM
Last modification on : Monday, March 30, 2020 - 1:50:03 PM
Long-term archiving on: : Thursday, March 30, 2017 - 2:44:21 PM

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  • HAL Id : tel-00621192, version 1

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Alaa Abdine. Développement et applications de protocoles de synthèse in vitro du canal mécanosensible bactérien à large conductance, pour son étude structurale par résonance magnétique nucléaire en phase solide. Sciences pharmaceutiques. Université Paris-Diderot - Paris VII, 2011. Français. ⟨tel-00621192⟩

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