Abstract
The function of the molecular chaperone Hsp90 depends on large conformational changes, the rearrangement of local motifs, and the binding and hydrolysis of ATP. The size and complexity of the Hsp90 system impedes the detailed investigation of their interplay using standard methods. To overcome this limitation, we developed a three-color single-molecule FRET assay to study the interaction of Hsp90 with a fluorescently labeled reporter nucleotide in detail. It allows us to directly observe the cooperativity between the two nucleotide binding pockets in the protein dimer. Furthermore, our approach disentangles the protein conformation and the nucleotide binding state of Hsp90 and extracts the kinetics of the state transitions. Thereby, we can identify the kinetic causes mediating the cooperativity. We find that the presence of the first nucleotide prolongs the binding of the second nucleotide to Hsp90. In addition, we observe changes in the kinetics for both the open and the closed conformation of Hsp90 in dependence on the number of occupied nucleotide binding sites. Our analysis also reveals how the co-chaperone Aha1, known to accelerate Hsp90's ATPase activity, affects those transitions in a nucleotide-dependent and independent manner, thereby adding another layer of regulation to Hsp90.