Keynote
Understanding Microsecond Functional Dynamics of Proteins with Single-Molecule Fluorescence Spectroscopy

The catalytic mechanisms of complex biological machines may involve a combination of chemical steps and conformational transitions. The latter are oftentimes hidden to traditional biochemical investigations, but can be exposed by single-molecule fluorescence experiments. Single-molecule FRET (smFRET) is an ideal tool to probe the conformational dynamics accompanying functional dynamics of biological machines. We recently developed a novel photon-by-photon analysis method for smFRET that facilitates studying conformational dynamics even on the microsecond time scale.

We first applied our new method to the domain closure reaction of the enzyme adenylate kinase. Surprisingly, we found that the bound enzyme opens and closes its domains much faster than the unbound enzyme, and two orders of magnitudes faster than the turnover rate of the enzyme! This exciting finding, which radically deviates from previous observations on adenylate kinase, led us to suggest that enzymes use numerous cycles of conformational rearrangement as a means to optimize the mutual orientation of their substrates for reaction.

Our photon-by-photon analysis method was also applied to study the functional dynamics of the disaggregating machine ClpB. This machine is comprised of six identical subunits arranged as a barrel. A coiled-coil domain resides on the outside surface of each subunit, and this domain has been implicated as a control element of the machine, to which the co-chaperone DnaK binds. We found that the coiled-coil domain populates several conformational states, which could be related some of these conformational states to well-known functional states of the machine. However, in contrast to the static picture arising from electron microscopy of ClpB, our data shows that the conformational states of the coiled-coil domain are interchanging on a sub-millisecond time scale. Binding of DnaK stabilizes the domain in its active form.