α-helix peptide folding and unfolding activation barriers:: A nanosecond UV resonance raman study

We used UV resonance Raman spectroscopy to characterize the equilibrium conformation and the kinetics of thermal denaturation of a 21 amino acid, mainly alanine, alpha-helical peptide (AP). The 204-nm UV resonance Raman spectra show selective enhancements of the amide vibrations, whose intensities and frequencies strongly depend on the peptide secondary structure. These AP Raman spectra were accurately modeled by a linear combination of the temperature-dependent Raman spectra of the pure random coil and the pure alpha-helix conformations; this demonstrates that the AP helix-coil equilibrium is well-described by a two-state model. We constructed a new transient UV resonance Raman spectrometer and developed the necessary methodologies to measure the nanosecond relaxation of AP following a 3-ns T-jump. We obtained the T-jump by using a 1.9-mu m IR pulse that heats the solvent water. We probed the AP relaxation using delayed 204-nm excitation pulses which excite the Raman spectra of the amide backbone vibrations. We observe little AP structural changes within the first 40 ns, after which the alpha-helix starts unfolding. We determined the temperature dependence of the folding and unfolding rates and found that the unfolding rate constants show Arrhenius-type behavior with an apparent NX kcal/mol activation barrier and a reciprocal rate constant of 240 +/- 60 ns at 37 degrees C. However, the folding rate constants show a negative activation barrier, indicating a failure of transition-state theory in the simple two-state modeling of AP thermal unfolding,which assumes a temperature-independent potential energy profile along the reaction coordinate. Our measurements of the initial steps in the or-helical structure evolution support recent protein folding landscape and funnel theories; our temperature-dependent rate constants sense the energy landscape complexity at the earliest stages of folding and unfolding.