Pyrene Excimer Formation (PEF) and Its Application to the Study of Polypeptide Dynamics

This Perspective describes how the fluorescence blob model (FBM) has been developed and applied over the past 30 years to characterize the long-range backbone dynamics (LRBD) of polymers in solution. In these experiments, the polymers are randomly labeled with the dye pyrene, which forms an excimer upon the encounter between an excited and a ground-state pyrenyl label inside a finite subvolume of the polymer coil referred to as a blob representing the volume probed by the excited pyrene. By compartmentalizing the polymer coil into a cluster of identical blobs, FBM analysis of the fluorescence decays acquired with the polymers yields the number N_blob of structural units inside a blob. Since a flexible or rigid backbone will result in an N_blob that is either large or small, N_blob can be used as a measure of the flexibility of a given polymer. After having established that these experiments based on pyrene excimer formation (PEF) yielded quantitative information about the LRBD of a variety of polymers in solution, control experiments were carried out to characterize the effects that different molecular variables, such as the side-chain size (SCS) of a structural unit or the length of the linker connecting pyrene to the polymeric backbone, had on the parameters retrieved with the FBM. At this point, the FBM was applied to study the LRBD of polypeptides prepared from racemic mixtures of amino acids (aa's). These studies led to the establishment of simple rules that could be developed into mathematical equations to describe the LRBD of polypeptides. The N_blob values retrieved from the FBM analysis of the fluorescence decays acquired with the pyrene-labeled polypeptides could then be employed to predict the total conformational search time (τ_tcs) of any polypeptide based on their sequence. Strong correlations were found between the predicted τ_tcs and the experimental folding times of 145 proteins. The good quality of these correlations suggests that the blob-based approach described in this report might represent an interesting mathematical means for studying protein folding.