Multiple proteins function as nanomachines, alternating chemical steps with conformational transitions. Single-molecule FRET (smFRET) spectroscopy is a powerful tool to study the internal motions of protein machines during function. We developed a novel photon-by-photon maximum likelihood algorithm for the analysis of smFRET experiments, which can track conformational dynamics even on the microsecond time scale. We used this algorithm to study several protein machines, finding in all cases surprisingly fast function-related dynamics.<br/>We first measured domain closing and opening times of the enzyme adenylate kinase, and found them to be two orders of magnitude shorter than the catalytic turnover time. We proposed that the enzyme uses these fast motions to optimize substrate orientation (1). We then studied internal dynamics of the large hexameric AAA+ machine ClpB, which rescues aggregated proteins by threading them through its central pore. The pattern of the microsecond dynamics of the pore loops, which protrude into the central pore and pull in substrate proteins, suggested a Brownian ratchet mechanism for protein threading (2). Finally, we studied the chaperone GroEL, which undergoes a series of conformational transitions between global allosteric states. The population ratio of four fast-exchanging microsates was found to determine the particular global state of the chaperone.<br/>Thus, it seems that the proteins we have probed can be described well as possessing a mode of action based on two time scales. The activity cycle of each of these proteins defines a relatively ‘slow’ time scale, limited by a ‘timer’ event such as ATP hydrolysis or product release. On the other hand, their structural fluctuations constitute a second, much faster time scale, which is employed by the protein to affect the slow activity cycle.

<b>Award Address</b> (Nakanishi Prize sponsored by the Nakanishi Prize Endowment). Two-time-scale paradigm for protein functional dynamics: Lessons from single-molecule FRET

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Award Address (Nakanishi Prize sponsored by the Nakanishi Prize Endowment). Two-time-scale paradigm for protein functional dynamics: Lessons from single-molecule FRET