Ecole Normale Supérieure, Paris, France
Department of chemistry
PhD thesis in the NMR team of Bodenhausen/Tekely/Abergel/Duma
Internal motions of proteins probed by solid-state NMR under magic-angle
spinning (MAS)
In the past decades, solution-state Nuclear Magnetic Resonance (NMR)
spectroscopy in liquids has evolved to become a powerful method for
the study of internal mobility in proteins and other biomolecules,
primarily through the measurement of spin relaxation rates and
residual dipolar couplings. These allow one to probe internal motions
occurring on different time scales, from picoseconds to milliseconds.
Moreover, structural biology has demonstrated the fundamental role of
the 3D structure of proteins to understand their biological function
at a molecular level. In addition to this well documented aspect,
recent studies have shown the importance of internal dynamics for
molecular interactions. This adds a third dimension to the classical
structure/function relationships, resulting in the more complex
structure/dynamics/function relationship.
Recent progress in solid-state NMR has opened the way to
investigations of proteins in microcrystalline form. It has become
possible to determine protein structures in the solid state. In
addition, recent observations suggest that internal dynamics of
microcrystalline proteins can also be accessed. In particular,
nitrogen-15 spin relaxation rates have been measured in small
proteins. This demonstrates the existence of motional processes on
time scales that are significantly longer than those accessible by NMR
relaxation measurements in solution.
The aim of this thesis is to explore the different ways for carrying
out successfully the study of internal dynamics in micro-crystalline
proteins by solid-state NMR. The proposed topic is a natural extension
of the research developed in our laboratory, namely the study of
internal dynamics of proteins in solution by NMR, and represents a
fundamental step towards the transition to a new spectroscopy allowing
the study dynamics of those systems, which cannot crystallize or be
studied in solution, i.e. solid-state NMR. First, the candidate will
endeavour to prepare the biologically relevant samples necessary for
probing dynamics by solid-state NMR either as micro-crystals or as a
micro-crystalline precipitate. Based on promising methods that are
currently under development in our laboratory, the candidate must
participate to the implementation of new approaches to the study of
proteins in the solid state. Another objective is the application of
these new methodologies to the study of the internal dynamics of the
human protein Centrin 2 (HsCen2) in micro-crystalline form. This
proposal also aims at characterizing the internal dynamics in the
complex formed by the C-terminal domain of this protein with a target
peptide (P17-XPC). Such studies involve protein expression,
purification and crystallisation, prior to the implementation of the
solid-state NMR methodologies for probing internal dynamics. Finally,
relying on numerical methods implemented recently in the host
laboratory, the candidate will further develop and adapt the model of
Network of Coupled Rotators to predict and interpret NMR relaxation
parameters observed in solids.
This PhD project will be conducted in close collaboration with
partners at CEA and Curie Institute. Strong knowledge in
biochemistry and crystallography will be highly valued but not
mandatory. Passion and interest for the combined
experimental/numerical approaches are essential. The thesis
will be supported by a grant that has been awarded by the
French Agence Nationale de la Recherche (ANR)
