Energy conversion of E. coli Hsp90 and yeast Hsp90

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The molecular chaperone Hsp90 is involved in folding, activation and stabilization of various substrate proteins. Its function is dependent on ATP binding and ATP hydrolysis. Recently published crystal structures of Hsp90 show a very good static picture and suggest large conformational changes during the ATPase cycle. We developed an assay that allows following the conformational dynamics of single Hsp90 dimers in real time under near physiological conditions. This is done with single molecule fluorescence resonance energy transfer (smFRET) and specifically labelled single-cystein mutants, which are specifically immobilized in microfluidic chambers. First experiments allowed us to observe the large conformational changes of single Hsp90 molecules in real time in a nucleotide dependent manner. The obtained dwell time distributions enabled us to propose a kinetic cycle for the opening and closing of Hsp90 without substrate and co-chaperones. The aim of this project is first to link these conformational changes to the chemical cycle of Hsp90 with the help of various (fluorescent) ATP analogues. Then, substrate and co-chaperone binding will be correlated with the conformational cycle of Hsp90. Finally, we will gain new insight into the function of tumor drugs that influence Hsp90 activity.