Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using temperature-jump IR spectroscopy and isotopic labeling.

Two antiparallel beta-strands connected by a turn make beta-hairpins an ideal model system to analyze the interactions and dynamics of beta-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form well-characterized beta-hairpin conformations in aqueous solution. Selective introduction of 13C=O groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I' band. These frequency patterns reflect theoretical predictions, and the coupled 13C=O band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of approximately 10 degrees C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of beta-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I' components. Analysis of the data supports multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding.