Single Molecule Fluorescence Methods for Molecular Chaperones and Their Client Interactions

Abstract

Single molecule fluorescence-based methods have significantly contributed to our understanding of molecular motors, protein–nucleic acid interactions and intrinsically disordered proteins. Their application to molecular chaperones is promising to understand multi-protein interactions but is still limited, largely due to low affinities between many chaperones and their clients (substrates). Here we summarize the state of the art for investigating chaperone dynamics and its regulation with a focus on multi-protein interactions. We review the timescales of conformational rearrangements of chaperones such as Hsp90 and Hsp70 in the presence of nucleotides and cochaperones. A combination of single molecule techniques such as smFRET, FCS and time-resolved anisotropy elucidates a dynamic picture of chaperones across timescales ranging from seconds to nanoseconds. Likely all these timescales play an important role in regulating the chaperone function. Therefore, such experimental approaches will further improve our fundamental understanding of chaperones and their client interactions.