Soutenance - Institut de Biologie Structurale

Soutenance
THESE
Mercredi 18 janvier 2017 à 14h
Salle des séminaires
Institut de biologie structurale - 71 avenue des Martyrs CS 10090 38044 Grenoble Cedex 9 - T.+33 (0)4 57 42 85 00
www.ibs.fr
par Vilius Kurauskas
Institut de Biologie Structurale
Groupe de de RMN biomoléculaire
The function of a membrane protein:
studies of structure and dynamics by NMR
Thèse de Doctorat de l’Université de Grenoble
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 μs dynamics in a crystalline ubiquitin to gain the insight on the impact of the crystalline lattice on such motions, using solid-state NMR and μs long MD simulations of explicit
crystal arrangements. Interestingly a local dynamic exchange process on a μs 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-μs time
scale, and provide evidence that overall and local motion are coupled. We discuss the implications of μs
dynamics on the data quality in X-ray diffraction experiments.
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