Heterogeneity in the Folding of Villin Headpiece Subdomain HP36

• Type of Material: Article
• Authors: Sureshbabu Nagarajan, Emory University; Shifeng Xiao, Shenzhen University; Daniel P. Raleigh, State University of New York; Brian Dyer, Emory University
• Language: English
• Date: 2018-12-13
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2018 American Chemical Society.
• Final Published Version (URL): http://dx.doi.org/10.1021/acs.jpcb.8b07683
• Title of Journal or Parent Work: Journal of Physical Chemistry B
• ISSN: 1520-6106
• Volume: 122
• Issue: 49
• Start Page: 11640
• End Page: 11648
• Grant/Funding Information: This work was supported by a NIH Grant GM053640 (R.B.D.) ;and NSF Grant MCB-1330259 (D.P.R.).
• Keywords: SIMULATIONS; Chemistry; COOPERATIVITY; Physical Sciences; DYNAMICS; ULTRAFAST; Science & Technology; MODEL; FAST EVENTS; TRANSITION-STATE; PROTEIN; Chemistry, Physical; KINETICS; EDITED IR
• Research Categories: Biology, Molecular; Chemistry, Physical

Abstract

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 13 Câ• 18 O 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, 13 C= 18 O 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 13 C= 18 O label by temperature dependent FTIR and temperature jump IR spectroscopy, respectively. Equilibrium experiments reveal that the 13 C= 18 O labels at different positions in HP36 show drastic differences in the midpoint of their transitions (T m ), 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 13 C= 18 O A57 is 2.4e + 05 s -1 whereas for 13 C= 18 O 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.