Probing Protein-Chaperone Interactions with Single-Molecule Fluorescence Spectroscopy

摘要

Molecular chaperones are an essential part of the cellular machinery that aids protein folding and assembly in vivo. Particularly remarkable are the members of the Hsp60 class, which encapsulate the folding protein in a central, closed cavity; the most well-studied example is the bacterial GroEL/ ES system. Work of the past two decades has resolved many aspects of the processes involved. However, remarkably little is known about the influence of the chaperone on the conformational distributions and folding mechanisms of its substrate proteins. Because of the structural heterogeneity of the nonnative substrate bound to a molecular machine in the 10~6 Da range, its experimental investigation has been difficult with established ensemble methods. Since single-molecule spectroscopy, in particular in combination with Forster resonance energy transfer (FRET), can provide distance and orientational information free of ensemble averaging and allows intramolecular distance dynamics to be observed at equilibrium, it is a promising approach to address such questions. Herein, we show how single molecule FRET can be utilized to investigate the nonnative conformation and dynamics of bovine rhodanese, a classic chaperone substrate protein, upon interaction with GroEL.