Single-molecule studies of protein folding

Although protein-folding studies began several decades ago, it is only recently that the tools to analyze protein folding at the single-molecule level have been developed. Advances in single-molecule fluorescence and force spectroscopy techniques allow investigation of the folding and dynamics of single protein molecules, both at equilibrium and as they fold and unfold. The experiments are far from simple, however, both in execution and in interpretation of the results. In this review, we discuss some of the highlights of the work so far and concentrate on cases where comparisons with the classical experiments can be made. We conclude that, although there have been relatively few startling insights from single-molecule studies, the rapid progress that has been made suggests that these experiments have significant potential to advance our understanding of protein folding. In particular, new techniques offer the possibility to explore regions of the energy landscape that are inaccessible to classical ensemble measurements and, perhaps, to observe rare events undetectable by other means.