PROTEIN DYNAMICS · Conformational Dynamics of Proteins by Solid-State NMR

Conformational Dynamics of Proteins by Solid-State NMR

The focus of the PIRG03-GA-2008-231026 "Protein Dynamics" project was development and application of new methods for probing molecular motions in proteins in the solid state. During the fellowship we have achieved most of our objectives regarding developing novel methodology. In collaboration with Martin Blackledge group from IBS, Grenoble we introduced a new model for interpreting 15N spin-lattice relaxation in the context of Anisotropic Collective Motions (ACM). Such motions are of particular scientific interest since they are often linked to functional motions in crucial biophysical processes such as enzymatic catalysis, molecular recognition, signaling, ligand binding, and protein folding. The model was described in a Journal of American Chemical Society publication on the example of collective motions of monomers in a protein dimer Crh (J. Am. Chem. Soc. 2010, 132, 1246-8). In collaboration with Martin Blackledge of IBS, Grenoble and Stephan Grzesiek of Biozentrum in Basel, we have also designed and validated an approach for measuring 13C spin-lattice relaxation rates in [U-13C, 15N] labeled proteins and thus achieved the first of the postulated milestones. More specifically, we have demonstrated that at spinning frequencies = < 60 kHz proton driven spin diffusion that usually leads to averaging of 13C longitudinal relaxation rates is sufficiently reduced to allow for site-specific relaxation measurements with a negligible or small error. The feasibility of the method was demonstrated on the protein GB1 providing a venue for extensive characterisation of both backbone and side-chain dynamics of proteins in the solid state. This study was published in the Journal of American Chemical Society (J. Am. Chem. Soc. 2010, 132, 8252-4.) Also in collaboration with Blackledge and Grzesiek we have developed an approach to probe slow molecular motions achieving the second milestone. We have shown that at spinning frequencies < 45 kHz the coherent contribution to the 15N R1rho becomes negligible and that the measurement reports primarily on the timescale and amplitude of slow motions even in fully protonated systems. The results of this study were described in the Journal of American Chemical Society (J. Am. Chem. Soc. 2011, 133, 16762-16765.). Thanks to the newly developed methods the increase of the available data points allowed us to consider more sophisticated models of molecular motions in proteins. The proposed approach may have wide implications for understanding dynamical transformations of proteins. For the protein GB1 we obtained 15N R1 rates at 5 magnetic fields from 600 to 1000 MHz, 15N R1rho rates at 2 magnetic fields, 13C'R1 rates at three magnetic fields from 500 to 1000 MHz, and 13Calpha and side-chain 13C at 800 MHz. The publication describing anisotropic motions in this protein based on these extensive data sets is currently under preparation. As a part of another milestone we undertook temperature dependent studies in the 100-300 K range on a nanocrystalline protein. The findings of this study contrasted against similar studies performed using neutron diffractions are a topic of another manuscript under preparation. Besides the main track of the fellowship the fellow was also part time engaged in other endeavors that resulted in a number of publications (see Section 2A).

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