Osmolyte-induced separation of the mechanical folding phases of ubiquitin.

Solvent molecules play key roles in the conformational dynamics of proteins. Here we use single molecule force-clamp spectroscopy to probe the role played by the stabilizing osmolyte glycerol on the conformational ensembles visited by a single ubiquitin protein folding after mechanical extension. Using a variety of force-pulse protocols, we find that glycerol stabilizes the native state of ubiquitin, making it more resistant to mechanical unfolding. We also find that although glycerol enhanced the hydrophobic collapse of unfolded and highly extended ubiquitins, it had no effect on the resulting collapsed states that are essential precursors of the folded state. These disparate effects of glycerol may be the result of distinct structural roles played by solvent molecules at the transition state of each folding ensemble. Our results open the way for a detailed analysis of the transition state structures that form along the folding trajectory of a mechanically extended protein.