The function of a membrane protein : studies of structure and dynamics by NMR

Abstract : The use of detergents is often unavoidable in the structural studies of membrane proteins. Dodecylphosphocholine (DPC) is one of the most commonly used detergents for such studies in solution state NMR spectroscopy. The effect of detergent on structure and dynamics remains an important and poorly understood question. In this study we have investigated millisecond dynamics, substrate binding and structural features of three different yeast proteins from mitochondrial carrier family (GGC1, ORC1 and AAC3) in DPC micelles. We have detected millisecond dynamics, which are asymmetrically distributed across the structure. Contrary to previous claims, we show that these dynamics are unrelated to function, as they are not affected by the substitutions which abolish mitochondrial carrier transport in proteoliposomes. Furthermore, we could show that the very well-defined substrate specificity of these proteins in membranes is abolished when they are reconstituted in DPC, questioning their functionality. Structural investigations have revealed that both tertiary and secondary structures of these carriers are perturbed in DPC micelles, with some TM helices showing substantial solvent exposure. We have concluded from these observations that DPC detergent strongly perturbs these, and likely other mitochondrial carriers by rendering them very flexible. Our findings point to a possibly general effect of this detergent on membrane proteins, as we discuss with examples of previously studied membrane proteins. In the second part we have addressed a fundamental question of protein dynamics: how do proteins move inside crystals? We have investigated ms dynamics in a crystalline ubiquitin to gain the insight on the impact of the crystalline lattice on such motions, using solid-state NMR and ms long MD simulations of explicit crystal arrangements. Interestingly a local dynamic exchange process on a ms time scale is still present in crystals. However, by comparing different crystal forms we establish that the thermodynamics of the exchanging states and their interconversion rate constants are significantly altered by the crystal contacts. Furthermore, we detect overall "rocking" motion of molecules in the crystal, occurring on a tens-of-ms time scale, and provide evidence that overall and local motion are coupled. We discuss the implications of ms dynamics on the data quality in X-ray diffraction experiments.
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Submitted on : Tuesday, January 16, 2018 - 11:22:52 AM
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Vilius Kurauskas. The function of a membrane protein : studies of structure and dynamics by NMR. Biomolecules [q-bio.BM]. Université Grenoble Alpes, 2017. English. ⟨NNT : 2017GREAV005⟩. ⟨tel-01685239⟩

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