Date : 20/04/2009

Laboratory
Laboratoire de RMN
ENS/UPMC/CNRS
Departement de Chimie
Ecole Normale Superieure
24, rue Lhomnd
75231 Paris Cedex 05
Director UMR 7203 - Solange Lavielle

PhD Supervisor
Geoffrey Bodenhausen
email : This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
phone : +33 1 44 32 33 89

Subjects / Tools-Methodologies:
1 : Experimental Protein Dynamics / Solid-State NMR Spectroscopy
2 :Theoretical Protein Dynamics / Computational (non-MD) Approaches
3 : Protein Crystallization / Typical Biochemical Techniques

Summary of lab's interests

The primary interest of the lab is the investigation of the dynamics of proteins. Our experimental approaches rely on solution- and solid-state Nuclear Magnetic Resonance (NMR)spectroscopy. We are very active in the field of NMR methodology and have developed a host of methods to characterize many dynamic effects over a wide range of time scales: from sub-nanosecond motions of protein backbones and side-chains to acide-base reactions and conformational exchange on micro-, millisecond and second timescales. We have also developed theoretical models employed to predict and analyze fast (sub-ns to several ns) motions in proteins. Our experimental and computational approaches are often tested on well-known proteins for validation and then used to unravel the molecular mechanisms underlying the function of proteins involved in important biological processes (Human Centrin and DNA repair; Engrailed 2 and development; T. brucei 6-PGL, a potential target against sleeping sickness).

Summary of project

In the past decades, solution-state Nuclear Magnetic Resonance (NMR) spectroscopy 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 micro-crystalline 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 get crystals 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 solids-state NMR either as micro-crystals or as a micro-crystalline precipitate. Based on promising methods that are currently under development in the host 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.