Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2

We report single-molecule folding studies of a small, single-domain protein , chymotrypsin inhibitor 2 (CI2). CI2 is an excellent model system for prot ein folding studies and has been extensively studied, both experimentally ( at the ensemble level) and theoretically. Conformationally assisted ligatio n methodology was used to synthesize the proteins and site-specifically lab el them with donor and acceptor dyes. Folded and denatured subpopulations w ere observed by fluorescence resonance energy transfer (FRET) measurements on freely diffusing single protein molecules. Properties of these subpopula tions were directly monitored as a function of guanidinium chloride concent ration. It is shown that new information about different aspects of the pro tein folding reaction can be extracted from such subpopulation properties. Shifts in the mean transfer efficiencies are discussed, FRET efficiency dis tributions are translated into potentials, and denaturation curves are dire ctly plotted from the areas of the FRET peaks. Changes in stability caused by mutation also are measured by comparing pseudo wild-type CI2 with a dest abilized mutant (K17G). Current limitations and future possibilities and pr ospects for single-pair FRET protein folding investigations are discussed.