Heterogeneity in the Folding of Villin Headpiece Subdomain HP36.

Small single domain proteins that fold on the microsecond time scale have been the subject of intense interest as models for probing the complexity of folding energy landscapes. The villin headpiece subdomain (HP36) has been extensively studied because of its simple three helix structure, ultrafast folding lifetime of a few microseconds, and stable native fold. We have previously shown that folding as measured by a single 13C═18O isotopic label on residue A57 in helix 2 occurs at a different rate than that measured by global probes of folding, indicating noncooperative complexity in the folding of HP36. In order to determine whether this complexity reflects intermediates or parallel pathways over a small activation barrier, 13C═18O labels were individually incorporated at six different positions in HP36, including into all 3 helices. The equilibrium thermal unfolding transitions and the folding/unfolding dynamics were monitored using the unique IR signature of the 13C═18O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the 13C═18O labels at different positions in HP36 show drastic differences in the midpoint of their transitions ( Tm), ranging from 45 to 67 °C. Heterogeneity is also observed in the relaxation kinetics; there are differences in the microsecond phase when different labeled positions are probed. At a final temperature of 45 °C, the relaxation rate for 13C═18O A57 is 2.4e + 05 s-1 whereas for 13C═18O L69 HP36 the relaxation rate is 5.1e + 05 s-1, two times faster. The observation of site-dependent midpoints for the equilibrium unfolding transitions and differences in the relaxation rates of the labeled positions enables us to probe the progressive accumulation of the folded structure, providing insight into the microscopic details of the folding mechanism.