Direct UV Raman Monitoring of 310-Helix and π-Bulge Premelting during r-Helix Unfolding

We used UV resonance Raman (UVRR) spectroscopy exciting at ∼200 nm within the peptide bond π f π* transitions to selectively study the amide vibrations of peptide bonds during R-helix melting. The dependence of the amide frequencies on theirΨ Ramachandran angles and hydrogen bonding enables us, for the first time, to experimentally determine the temperature dependence of the peptide bond Ψ Ramachandran angle population distribution of a 21-residue mainly alanine peptide. TheseΨ distributions allow us to easily discriminate between R-helix, 310-helix and π-helix/bulge conformations, obtain their individual melting curves, and estimate the corresponding Zimm and Bragg parameters. A striking finding is that R-helix melting is more cooperative and shows a higher melting temperature than previously erroneously observed. TheseΨ distributions also enable the experimental determination of the Gibbs free energy landscape along the Ψ reaction coordinate, which further allows us to estimate the free energy barriers along the AP melting pathway. These results will serve as a benchmark for the numerous untested theoretical studies of protein and peptide folding.