Direct UV Resonance Raman Monitoring of Protein Folding Reaction Coordinate: alpha-Helix Melting and Formation Revisited

Spectroscopic investigations of protein folding mechanism(s) require the elucidation of the relationship between spectral features and the protein folding reaction coordinate(s). This thesis describes the development and demonstrates the applications of a novel UV resonance Raman (UVRR) spectroscopic methodology, which quantitatively correlates the UVRR amide III3 (AmIII3) frequency of a peptide bond to its Psi Ramachandran angle (which is arguably the most important folding reaction coordinate). This information, then, allows us, for the first time, to obtain Psi angular population distributions of peptide bonds in peptides and proteins from their UVRR AmIII3 band profiles, as well as the Gibbs free energy landscapes along the Psi Ramachandran angle folding coordinate. Application of this methodology allows us to quantitatively characterize the ensembles of folded and unfolded states in peptides and proteins. We show that the unfolded state ensembles show no evidence of completely disordered "random coil" conformation, and are usually dominated by PPII-like conformations. In addition, we for the first time experimentally detected the extended 2.51-helix conformation in poly-L-lysine and poly-L-glutamic acid, which is stabilized by electrostatic repulsion between side-chain charges. Most importantly, we resolved and quantitatively characterized the 310-helix and pi-bulge contributions within the "alpha-helix melting" of ala-rich peptides. We for the first time obtained their individual experimental melting curves, estimated their Zimm and Bragg parameters, and estimated their individual kinetic (un)folding rates. These results challenge the classical view of protein folding and provide an important quantitative basis for future studies