Native state conformational heterogeneity of HP35 revealed by time-resolved FRET.

ABSTRACT: The villin headpiece subdomain (HP35) has become one of the most widely used model systems in protein folding studies, due to its small size and ultrafast folding kinetics. Here, we use HP35 as a test bed to show that the fluorescence decay kinetics of an unnatural amino acid, p-cyanophenylalanine (Phe(CN)), which are modulated by a nearby quencher (e.g., tryptophan or 7-azatryptophan) through the mechanism of fluorescence resonance energy transfer (FRET), can be used to detect protein conformational heterogeneity. This method is based on the notion that protein conformations having different donor-acceptor distances and interconverting slowly compared to the fluorescence lifetime of the donor (Phe(CN)) would exhibit different donor fluorescence lifetimes. Our results provide strong eviden

SUBMITTER: Serrano AL 

PROVIDER: S-EPMC3438901 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature