Probing the folding and unfolding dynamics of secondary and tertiary structures in a three-helix bundle protein.

Fast relaxation kinetics studies of the B-domain of staphylococcal protein A were performed to characterize the folding and unfolding of this small three-helix bundle protein. The relaxation kinetics were initiated using a laser-induced temperature jump and probed using time-resolved infrared spectroscopy. The kinetics monitored within the amide I' absorbance of the polypeptide backbone exhibit two distinct kinetics phases with nanosecond and microsecond relaxation times. The fast kinetics relaxation time is close to the diffusion limits placed on protein folding reactions. The fast kinetics phase is dominated by the relaxation of the solvated helix (nu = 1632 cm(-1)), which reports on the fast relaxation of the individual helices. The slow kinetics phase follows the cooperative relaxation of the native helical bundle core that is monitored by both solvated (nu = 1632 cm(-1)) and buried helical IR bands (nu = 1652 cm(-1)). The folding rates of the slow kinetics phase calculated over an extended temperature range indicate that the core formation of this protein follows a pathway that is energetically downhill. The unfolding rates are much more strongly temperature-dependent indicating an activated process with a large energy barrier. These results provide significant insight into the primary process of protein folding and suggest that fast formation of helices can drive the folding of helical proteins.