Evidence of an intermediate and parallel pathways in protein unfolding from single-molecule fluorescence

Determining how proteins fold into their native structures is a subject of great importance, since ultimately it will allow protein structure and function to be predicted from primary sequence data. In addition, there is now a clear link between protein unfolding and misfolding events and many disease states. However, since proteins fold over rugged, multidimensional energy landscapes, this is a challenging experimental and theoretical problem. Single-molecule fluorescence methods developed over the past decade have the potential to follow the unfolding/folding of individual molecules. Mapping out the landscape without ensemble averaging will enable the identification of intermediate states which may not be significantly populated, in addition to the presence of multiple pathways. To date, there have been only a limited number of single-molecule folding/unfolding studies under nonequilibrium conditions and no intermediates have been observed. Here, for the first time, we present a single-molecule study of the unfolding of a large autofluorescent protein, Citrine, a variant of green fluorescent protein. Single-molecule fluorescence techniques are used to directly detect an intermediate on the unfolding/folding pathway and the existence of parallel unfolding pathways. This work, and the novel methods used, shows that single-molecule fluorescence can now provide new, hitherto experimentally inaccessible, insights into the folding/unfolding of proteins.