Exploring protein structure and dynamics under denaturing conditions by single-molecule FRET analysis

Proteins are highly complex biopolymers, exhibiting a substantial degree of structural variability in their properly folded, native state. In the presence of denaturants, this heterogeneity is greatly enhanced, and fluctuations take place among vast numbers of folded and unfolded conformations via many different pathways. To better understand protein folding it is necessary to explore the structural and energetic properties of the folded and unfolded polypeptide chain, as well as the trajectories along which the chain navigates through its multidimensional conformational energy landscape. In recent years, single-molecule fluorescence spectroscopy has been established as a powerful tool in this research area, as it allows one to monitor the structure and dynamics of individual polypeptide chains in real time with atomic scale resolution using Forster resonance energy transfer (FRET). Consequently, time trajectories of folding transitions can be directly observed, including transient intermediates that may exist along these pathways. Here we illustrate the power of single-molecule fluorescence with our recent work on the structure and dynamics of the small enzyme RNase H in the presence of the chemical denaturant guanidinium chloride (GdmCl). For FRET analysis, a pair or fluorescent dyes was attached to the enzyme at specific locations. In order to observe conformational changes of individual protein molecules for up to several hundred seconds, the proteins were immobilized on nanostructured, polymer coated glass surfaces specially developed to have negligible interactions with folded and unfolded proteins. The single-molecule FRET analysis gave insight into structural changes of the unfolded polypeptide chain in response to varying the denaturant concentration, and the time traces revealed stepwise transitions in the FRET levels, reflecting conformational dynamics. Barriers in the free energy landscape of RNase H were estimated from the kinetics of the transitions.