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

We used UV resonance Raman spectroscopy to characterize the equilibrium con formation and the kinetics of thermal denaturation of a 21 amino acid, main ly alanine, alpha-helical peptide (AP). The 204-nm UV resonance Raman spect ra show selective enhancements of the amide vibrations, whose intensities a nd frequencies strongly depend on the peptide secondary structure. These AP Raman spectra were accurately modeled by a linear combination of the tempe rature-dependent Raman spectra of the pure random coil and the pure alpha-h elix conformations; this demonstrates that the AP helix-coil equilibrium is well-described by a two-state model. We constructed a new transient UV res onance Raman spectrometer and developed the necessary methodologies to meas ure the nanosecond relaxation of AP following a 3-ns T-jump. We obtained th e T-jump by using a 1.9-mu m IR pulse that heats the solvent water. We prob ed the AP relaxation using delayed 204-nm excitation pulses which excite th e Raman spectra of the amide backbone vibrations. We observe little AP stru ctural changes within the first 40 ns, after which the alpha-helix starts u nfolding. We determined the temperature dependence of the folding and unfol ding rates and found that the unfolding rate constants show Arrhenius-type behavior with an apparent NX kcal/mol activation barrier and a reciprocal r ate constant of 240 +/- 60 ns at 37 degrees C. However, the folding rate co nstants show a negative activation barrier, indicating a failure of transit ion-state theory in the simple two-state modeling of AP thermal unfolding,w hich assumes a temperature-independent potential energy profile along the r eaction coordinate. Our measurements of the initial steps in the or-helical structure evolution support recent protein folding landscape and funnel th eories; our temperature-dependent rate constants sense the energy landscape complexity at the earliest stages of folding and unfolding.