Studying the Hsp90 Machinery in Living Cells by Single Molecule FRET

The 90 kDa heat shock protein Hsp90 is an ubiquitous molecular chaperone regulated by cochaperones, overexpressed upon stress and involved in several cellular processes such as proliferation, signal transduction and transcription. The interaction with various oncoproteins makes Hsp90 an attractive drug target. It remains unclear, how exactly Hsp90 fulfills its various roles within a cell and how drugs affect Hsp90 inside a cell.Single molecule Forster Resonance Energy Transfer (smFRET) has been used to study the mechanism of Hsp90's ATPase and chaperoning function. These experiments showed that the conformational dynamics of yeast Hsp90 is dominated by thermal fluctuations and not by nucleotides [1]. The presence of the cochaperone p23 increases the effect of nucleotides and introduces some directionality in Hsp90's ATPase cycle [2]. However, until now more than 20 cochaperones have been identified in eukaryotes, which makes “bottom up” in vitro experiment very difficult. Here we therefore study the dynamics of Hsp90 and the associated heavily regulated complex machinery [3] by applying smFRET on it in living cells.We build on previous approaches [4,5] and decrease background noise and increase observation time. This allows us to study Hsp90 in its native environment and likely to directly observe drug effects on their target molecule in a cell.[1] Ratzk et al. “Hsp90's mechano-chemical cycle is dominated by thermal fluctuations”, PNAS,2012.[2] Ratzke, et al. “Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery”, Nature Communications, 2014.[3] Taipale et al. “HSP90 at the hub of protein homeostasis: emerging mechanistic insights”, Nature Reviews, 2010.[4] Crawford, Torella, Aigrain, Plochowietz et al. “Long-lived intracellular single-molecule fluorescence using electroporated molecules”, Biophys J, 2013.[5] Konig et al. “Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells”, Nature Methods, 2015.