Heat shock protein 90 (Hsp90) is a master chaperone, regulating processes involved in cell cycle control, protein folding, and degradation; however, it does not work alone. There are a large number of co-chaperones that are known to bind to Hsp90 and regulate its ATP hydrolysis and chaperoning activity. The most potent stimulator of Hsp90’s ATP hydrolysis activity is Aha1, named after its role as “activator of Hsp90 ATPase.” But how do Aha1 and Hsp90 interact in detail? We answered this question by using advanced single-molecule techniques, specifically single-molecule Förster resonance energy transfer and optical tweezers. Low affinities are the limiting factor in such experiments; we overcame them by creating a tethered construct, in which Aha1 was connected to Hsp90 via a long unstructured amino acid linker. We were able to see, among other things, that one single bound Aha1 is sufficient to trigger Hsp90 to transition from an open to a closed conformation, but that two Aha1 molecules are necessary to fully stimulate Hsp90’s ATPase activity.

To illustrate all the novel methodological aspects of our paper, the cover image of the September 5 issue of Biophysical Journal schematically illustrates our tethered Aha1-Hsp90 construct with attached fluorophores in an optical tweezers setup. This image was inspired by our experimental methods that allow us to “make the invisible visible,” i.e., observe nanoscale structural changes that occur below the optical diffraction limit, in real time, by using force and fluorescent labels. We can now understand how structural changes in proteins and their dynamics are important for protein function. This knowledge is not only crucial for a thorough understanding of protein interactions, but can also be important for the development of targeted therapeutics.

You can read more about our research in the Hugel lab here, and in the KT lab here.

—Tanumoy Mondol, Laura-Marie Silbermann, Julia Schimpf, Leonie Vollmar, Bianca Hermann, Katarzyna (Kasia) Tych, and Thorsten Hugel