Pyrene Excimer Fluorescence as a Direct and Easy Experimental Means To Characterize the Length Scale and Internal Dynamics of Polypeptide Foldons

Abstract

This report provides evidence that the blobs characterized through the application of the fluorescence blob model (FBM) to the analysis of the fluorescence decays acquired with pyrene-labeled polypeptides are equivalent to the foldons used in the study of protein folding. The FBM was applied to characterize the length and time scale over which pyrene excimer formation (PEF) took place between pyrene labels covalently attached onto α-helical and partially helical poly(l-glutamic acid) (Py-PLGA) in DMF and DMSO, respectively, and unfolded poly(dl-glutamic acid) (Py-PDLGA) in both DMF and DMSO. The blob size obtained for α-helical Py-PLGA in DMF and the characteristic time determined for the backbone dynamics of unfolded Py-PDLGA matched very closely the expected size of foldons and their characteristic folding times, respectively. In particular, the blob size was confirmed by conducting molecular mechanics optimizations (MMOs) with HyperChem. Furthermore, the level of pyrene clustering along the polypeptides correlated nicely with their expected conformation, either coiled or helical for the Py-PDLGA or Py-PLGA constructs in DMF, respectively. Consequently, these results suggest that PEF experiments conducted on pyrene-labeled polypeptides provide valuable information on the time and length scales experienced by the amino acids located inside a polypeptide blob and that if polypeptide blobs and foldons present similar length and time scales, both entities must be equivalent. Since dynamic or spatial information on foldons is usually retrieved by conducting NMR or hydrogen exchange mass spectrometry experiments, PEF might thus provide an alternative, possibly simpler, route toward the characterization of polypeptide foldons in solution.