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Etude de complexes protéiques non covalents par spectrométrie de masse FT-ICR.

Abstract : The outstanding analytical performances of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS) were applied to study non-covalent interactions in protein complexes. In a first part, the application of this technique to the detection of intact protein complexes was developed on the system of Creatine kinase (CK) and its ligands ADP and ATP, leading to the observation of these complexes in the gas phase. Specific and non- specific contributions of these interaction were separated by means of a statictical model, yielding interaction constants (K1,ADP=11.8 ± 1.5 μM, K2,ADP=48 ± 6 μM, K1,ATP=27 ± 7 μM, K2,ATP=114 ± 27 μM) that are in fair agreement with literature values. In a second part, methods to study the surface accessibility of protein complexes by chemical modification in solution, either reversible (H/D exchange) or irreversible (specific modification of His and Lys by DEPC) were developed and implemented. The modification site was identified either by a bottom-up (enzymatic digestion followed by mass measurement of the resulting peptides) or a top-down approach (gas phase fragmentation of the entire protein by electron capture dissociation [ECD]). Within the framework of the current instrumentation, the latter approach yields reasonable results for proteins as large as 17 kDa and should be extendible to 25 kDa. Application of the different analytical strategies yielded insight into the structure and conformational dynamics of prion protein (PrP) oligomers that were obtained by partial thermal denaturation of the monomeric species in vitro. Increase in deuterium incorporation in some regions of the oligomeric species (compared to the monomer) reflects partial unfolding of the protein, as indicated by the unfolding of an α helix.
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Submitted on : Tuesday, July 27, 2010 - 10:39:53 AM
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  • HAL Id : pastel-00002922, version 1



Thorsten Daubenfeld. Etude de complexes protéiques non covalents par spectrométrie de masse FT-ICR.. Chimie. Ecole Polytechnique X, 2006. Français. ⟨pastel-00002922⟩



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