Intrinsic dynamics of globular proteins : Precise elucidation of slow motional modes and their relation to function

Protein Dynamics by NMR

Determination of protein backbone dynamics from residual dipolar couplings

While solution NMR is now established as the method of choice for studying local dynamics on picosecond to nanosecond timescales, dynamics occurring on slower time-scales are also of particular interest because many biologically important processes, such as enzymatic catalysis, signal transduction, ligand binding and allosteric regulation are expected to occur in this range. In our group we use residual dipolar couplings, combined with advanced analytical and simulation methods, to develop a precise model of slower motions occurring in proteins.

Slow correlated motions in protein GB3

An extensive set of RDCs, in combination with a motional model providing for anisotropic peptide plane reorientation in three orthogonal dimensions, has been used to identify and cross-validate the presence of conformational dynamics in protein G. Comparison with the amplitude and position of rapid motions in the same protein reveals a heterogeneous distribution of slow dynamics in the nanosecond to millisecond time range. Slower motions occur in the loops, and in the beta-sheet, and are absent in other regions of the molecule, including the alpha-helix. In the beta-sheet an alternating pattern of dynamics along the peptide sequence is found to form a long-range network across the beta-strands. Analysis of scalar couplings across hydrogen bonds strongly suggests that the motion is correlated across, and propagated via inter-strand hydrogen bonds. Such clear evidence that dynamical information is transmitted across hydrogen bond networks carries important implications for understanding the mechanism of information transfer in proteins occurring in processes such as allosteric regulation.

The amplitude of the collective motion increases across the beta-sheet with the highest amplitude in external strand. The residues exhibiting the highest level of flexibility coincide precisely with the sites participating in the interaction of protein G with its physiological partner, the antigen binding domain of immunoglobulin G. We propose that molecular interaction is facilitated by the increased conformational sampling due to this collective motion.

The existence of these slow motional modes extending across the entire beta-sheet carries clear implications for understanding the mechanisms of long-range signal propagation in proteins. In the case of protein G, these findings illustrate how the protein harnesses thermal motions via specific dynamic networks to enable molecular function at the interaction site.

The interpretation of conformational dynamics requires a precise description of the mean conformation, and in principle NMR data encode both structural and dynamic aspects of conformational space. Combining revolutionary techniques developed in our group for the determination of protein structure using only RDCs with novel approaches to the interpretation of RDCs in terms of dynamic modes, we are currently developing methods to simultaneously determine both structure and dynamics occurring up to the millisecond.

Identification of slow correlated motions in proteins using residual dipolar and hydrogen-bond scalar couplings.G. Bouvignies, P. Bernado, S. Meier, K. Cho, S. Grzesiek, R. Bruschweiler and M. Blackledge Proc. Natl. Acad. Sci. 102, 13885-13890 (2005)

Simultaneous Determination of Protein Structure and Dynamics using Residual Dipolar Couplings. G. Bouvignies, R.Bruschweiler and M. Blackledge. J.Am.Chem.Soc.128, 15100-15101 (2006).

Characterization of Protein Dynamics from Residual Dipolar Couplings using the Three Dimensional Gaussian Axial Fluctuation Model. Guillaume Bouvignies, Phineus R. L. Markwick and Martin Blackledge Proteins: Structure, Function and Bioinformatics71, 353-363 (2008).

Intrinsic dynamics of globular proteins : Simulation of slower motional modes

We have thus developed approaches to study functionally important slower intrinsic motions in proteins directly from experimental data. However until now no molecular simulation methods have been available to aid in the interpretation of these data due to the very long time scale motions involved in the averaging mechanism. This situation has potentially dramatically changed with the development of accelerated molecular dynamics (AMD) approaches that provide enhanced access to rare conformational events. This opens up remarkable perspectives that we will continue to explore, to enhance our understanding of slower motional modes in proteins. We have applied enhanced sampling techniques for the first time to the identification of slow molecular motions detected using RDCs.

Exploring Multiple Timescale Motions in Protein GB3 using Accelerated Molecular Dynamics and NMR. P. Markwick, G. Bouvignies and M. Blackledge J.Am.Chem.Soc.129, 4724-4730 (2007).

Detailed description of intrinsic protein dynamics in the solid state.

In collaboration with Professor Emsley at the NMR centre in Lyon we are also developing methods to study intrinsic dynamics of the same molecular system in the solid state. Here we hope to lay the foundations for future studies of motion in all solid state proteins, including fibrillar or membrane phases.

The role of 15N CSA and CSA/dipole cross correlation in 15N relaxation in solid proteins. J. Sein, N. Giraud, M. Blackledge and L. Emsley. J. Magn. Reson. 186, 26-33 (2007)

The influence of nitrogen-15 proton-driven spin diffusion on the measurement of nitrogen-15 longitudinal relaxation times. N. Giraud, M.Blackledge, A.Bockmann and L.Emsley. J. Magn. Reson. 184, 51-61 (2007)

Observation of Heteronuclear Overhauser Effects Confirms the 15N-1H Dipolar Relaxation Mechanism in a Crystalline Protein. N. Giraud, J. Sein, G. Pintacuda, A. Bockmann, A. Lesage, M. Blackledge and L. Emsley J.Am.Chem.Soc. 128, 12398-12399 (2006).

Quantitative Analysis of Backbone Dynamics in a Crystalline Protein from Nitrogen-15 Spin-Lattice Relaxation. N. Giraud, M. Blackledge, M. Goldman, A. Bockmann, A. Lesage, F. Penin and L. Emsley. J.Am.Chem.Soc.127, 18190-18201 (2005).