Combining Single-Molecule Fluorescence and MD-Simulations to Delineate the Kinetics and Regulation of Proteins

Abstract

Protein conformational kinetics and its regulation occur on many time- and length-scales. While for example post-translational modifications act very locally, specific protein-protein interaction typically affect larger domains, and global changes (like crowding) affect the whole protein (complex) non-specifically. In a first part we use a combination of single-molecule FRET, FRET-FCS, ns-FCS and MD simulations to delineate the timescales for information transfer from the hydrolysis event in the nucleotide binding site of the heat shock protein 90 (Hsp90) to large conformational changes in the Hsp90 dimer[1]. This allosteric process occurs via a hierarchical mechanism that involves timescales from pico- to microseconds. In a second part we show protein regulation under different degrees of localization, and present the effects on the Hsp90 chaperone system. Interestingly using single-molecule FRET, we find that similar functional and conformational steady states are caused by completely different underlying kinetics. We will introduce a new mechanistic concept, namely functional stimulation through conformational confinement[2]. Finally, we use a network of distance distributions obtained with single molecule FRET to investigate a Hsp90 - model client interaction[3]. We find negligible effects of the client on the open-close equilibrium of Hsp90, but strong effects on Hsp90's conformational fluctuations. This hints towards an additional (orthogonal) regulation mechanism. Altogether, these applications demonstrate that a combination of (single-molecule) methods is ideal to investigate the biophysics of multi-component (protein) systems. [1] S. Wolf et al., Allosteric action of nucleotides on Hsp90 across several time- and length scales. BioRxiv, 2020.02.15.950725 (2020); https://doi.org/10.1101/2020.02.15.950725 [2] S. Schmid and T. Hugel, Controlling protein function by fine-tuning conformational flexibility. eLife, 9, 1013 (2020); https://doi.org/10.7554/eLife.57180 [3] Hellenkamp et al., Multidomain structure and correlated dynamics determined by self-consistent FRET networks. Nat Methods, 14 (2), 174 (2017). https://doi.org/10.1038/nmeth.4081