Direct UV Raman monitoring of 3(10)-helix and pi-bulge premelting during alpha-helix unfolding.
We used UV resonance Raman (UVRR) spectroscopy exciting at approximately 200 nm within the peptide bond pi --> pi* transitions to selectively study the amide vibrations of peptide bonds during alpha-helix melting. The dependence of the amide frequencies on their Psi Ramachandran angles and hydrogen bonding enables us, for the first time, to experimentally determine the temperature dependence of the peptide bond Psi Ramachandran angle population distribution of a 21-residue mainly alanine peptide. These Psi distributions allow us to easily discriminate between alpha-helix, 3(10)-helix and pi-helix/bulge conformations, obtain their individual melting curves, and estimate the corresponding Zimm and Bragg parameters. A striking finding is that alpha-helix melting is more cooperative and shows a higher melting temperature than previously erroneously observed. These Psi distributions also enable the experimental determination of the Gibbs free energy landscape along the Psi 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.
Mikhonin
Aleksandr V
AV
Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Asher
Sanford A
SA
D000577:Amides / Q000737:chemistry; D006860:Hydrogen Bonding; D010455:Peptides / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics
D000577:Amides; D010455:Peptides; D011506:Proteins