NMR STUDIES OF Protein DYNAMICS – from fundamental biophysics TO BIOLOGICAL function

Martin Blackledge

Protein Dynamics and Flexibility by NMR, Institut de Biologie Structurale, Grenoble

martin.blackledge@ibs.fr

The precision with which X-ray crystallography and electron microscopy provides structural models of biologically active protein conformations belies an easily overlooked dilemma. Proteins are inherently dynamic, exhibiting conformational freedom on many timescales,¹ implicating structural rearrangements that play a major role in molecular interaction, thermodynamic stability and molecular recognition and signalling. Static structural biology affords little insight into the nature of the underlying conformational equilibrium that is essential for biological function. NMR spectroscopy, in combination with single molecule FRET and ensemble scattering approaches, provides unique characterization of structural and dynamic properties averaged over all states sampled in solution.

Intrinsically disordered proteins (IDPs) represent extreme examples where flexibility defines molecular function. IDPs exhibit highly heterogeneous local and long-range structural and dynamic propensities, sampling a much flatter energy landscape than their folded counterparts, allowing inter-conversion between a quasi-continuum of accessible conformations (the determination of a single representative structure has no physical relevance). While considerable effort has been devoted to describe the conformational space sampled by IDPs,² little is known about the timescale of their intrinsic dynamics. Using temperature dependent NMR relaxation, we recently identified distinct local and long-range conformational dynamics in IDPs occurring on timescales covering three orders of magnitude.³

In spite of the ubiquitous nature of IDPs, the molecular mechanisms regulating their interactions with physiological partners remains poorly understood. We use NMR spectroscopy to map these complex molecular recognition trajectories, from the highly dynamic free-state equilibrium to the bound state ensemble. Examples include the replication machinery of paramyxoviruses, where the highly (>70%) disordered phosphoprotein initiates transcription and replication via its interaction with the disordered domain of the nucleoprotein,⁴ the JNK signalling pathway, where specificity is controlled by disordered domains of MAP kinases,⁵ or the nuclear pore, where weak interactions between the nuclear transporter and highly flexible chains containing multiple ultra-short recognition motifs, facilitate fast passage into the nucleus.⁶ Finally, a combination of solution techniques reveals large-scale domain dynamics in the C-terminus of Influenza H5N1 polymerase that are essential for import into the nucleus of the infected cell. ⁷

1. Lewandowski, Halse, Blackledge, Emsley 348, 578 Science (2015)
2. Jensen, Zweckstetter, Huang, Blackledge 114, 6632 Chem Rev (2014)
3. Abyzov, Salvi, Jensen, Ruigrok, Blackledge 138, 6240-6251 J. Am. Chem. Soc. (2016)
4. Schneider, Maurin, … Ruigrok, Jensen, Blackledge J. Am. Chem. Soc. 137,1220 (2015)
5. Kragelj, Palencia, Nanao, Maurin, Bouvignies, Blackledge, Jensen Proc. Natl. Acad. Sci. 112, 3409 (2015)
6. Milles, Mercadente …. Blackledge, Gräter, Lemke Cell 163, 734-745 (2015)
7. Delaforge, Milles…. Lemke, Hart, Blackledge J. Am. Chem. Soc. 137, 15122-15134 (2015)