Can microsecond domain motion affect enzymatic activity? A single-molecule FRET study

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Proteins are involved in a broad spectrum of functions, acting as enzymes, signal transductors and more. Many of them have evolved to harness largescale motions of domains and subunits to promote their functionality. Studying the structural dynamics of protein machines is essential for deciphering how they function and in what way they are regulated. The knowledge acquired through such studies is not only very relevant for engineering new proteins, it can also open unprecedented opportunities for the design of drugs.The accurate relation between conformational dynamics and the chemical steps of enzymatic catalysis has been debated extensively. In various enzymes, the active site is situated in a cleft between two domains, and structural rearrangements close the domains over the bound substrate. While current experimental techniques, from x-ray crystallography to cryo-electron microscopy, can readily observe the end points of such motions, measuring the associated time scales is often challenging. Single-molecule Förster resonance energy transfer (smFRET) spectroscopy can measure large-scale motions in real time, with the great advantage that multiple time scales can be covered. Combined with H2MM, a photon-by-photon hidden Markov model analysis, smFRET studies on the enzyme adenylate kinase (AK) have recently revealed very fast domain motions, two orders of magnitude faster than the turnover of the enzyme. AK may use numerous cycles of conformational rearrangement in order to find a relative orientation of its substrates that is optimal for their chemical reaction. Preliminary studies on the effect of urea on the substrate inhibition of AK are in support of this idea, indicating that a delicate balance between domain opening and closing is important for efficient turnover.However, it is not clear yet whether this behavior is specific to AK or applies to a multitude of enzymes, which requires expanding our studies to other proteins. In the enzyme phosphoglycerate kinase (PGK) the catalytic reaction is accompanied by a large-scale hinge-bending motion, rendering it a good target for this evaluation. We will ask: How fast are the motions? What is the relation between catalytic reaction and domain closure? Can the dynamics be described as jumps between two states or is a more complex molecular model required? Are motions within a domain also necessary in order to reach a fully closed state? The smFRET experiments we propose can provide a major contribution to clarify these issues. We aim to see a definitive correlation between the motions of the protein and its activity. Overall, we plan to not only characterize fast, sub-millisecond domain motions in PGK, but also establish a conclusive role for these motions in enzyme catalysis. This has the potential to modify our picture of the relation of dynamics and function dramatically.