α-Helix Peptide Folding and Unfolding Activation Barriers: A Nanosecond UV Resonance Raman Study

Abstract

We used UV resonance Raman spectroscopy to characterize the equilibrium conformation and the kinetics of thermal denaturation of a 21 amino acid, mainly alanine, R-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 R-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-μm 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 R-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 ∼8 kcal/mol activation barrier and a reciprocal rate constant of 240 ( 60 ns at 37 °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 R-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.