Site-specific conformational determination in thermal unfolding studies ofhelical peptides using vibrational circular dichroism with isotopic substitution

Understanding the detailed mechanism of protein folding requires dynamic, s ite-specific stereochemical information. The short time response of vibrati onal spectroscopies allows evaluation of the distribution of populations in rapid equilibrium as the peptide unfolds. Spectral shifts associated with isotopic labels along with local stereochemical sensitivity of vibrational circular dichroism (VCD) allow determination of the segment sequence of unf olding. For a series of alanine-rich peptides that form alpha-helices in aq ueous solution, we used isotopic labeling and VCD to demonstrate that the a lpha-helix noncooperatively unwinds from the ends with increasing temperatu re. For these blocked peptides, the C-terminal is frayed at 5 degrees C. Ab initio level theoretical simulations of the in and VCD band shapes are use d to analyze the spectra and to confirm the conformation of the labeled com ponents. The VCD signals associated with the labeled residues are amplified by coupling to the nonlabeled parts of the molecule. Thus small labeled se gments are detectable and stereochemically defined in moderately large pept ides in this report of site-specific peptide VCD conformational analysis.