Ultrafast Events in the Folding of Ferrocytochrome c † Rajesh Kumar, N. Prakash Prabhu, and Abani K. Bhuyan* School of Chemistry, UniVersity of Hyderabad, Hyderabad 500046, India ReceiVed March 1, 2005; ReVised Manuscript ReceiVed April 14, 2005 ABSTRACT: Laser flash photolysis and stopped-flow methods have been used to study the dynamic events in the micro- to millisecond time bin in the refolding of horse ferrocytochrome c in the full range of guanidine hydrochloride concentration at pH 12.8 ((0.1), 22 °C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 µs. The chain then undergoes diffusive dynamics-mediated contraction and expansion, in which intrapolypeptide ligands make transient contacts with the heme iron, giving rise to two distinct kinetic phases of ∼0.4 and ∼3 µs. Under moderate to absolute refolding conditions, the rates of these processes show little dependence on the denaturant concentration, indicating the absence of structural element in the incipient or the relaxed state. Chain expansion and contraction events continue until the polypeptide finds a stable and supportive transition state. The crossing of this transition barrier, which rate-limits the folding of alkaline ferrocytochrome c, is characterized by a stopped-flow measured time constant of ∼3 ms in aqueous solvent. Observed kinetics thus implicate no submillisecond folding structure. The folding kinetics is effectively two state in which the unfolded polypeptide first relaxes to an unstructured chain and then crosses over a late rate-limiting barrier to achieve the native conformation. The experimentally observed rates as a function of guanidine hydrochloride concentration have been simulated by numerically calculated microscopic rates of a simple kinetic model that captures the essential features of folding. Isolated peptide fragments in solution often show a tendency to form helices and turns (1-3), and they may form too rapidly in the intact protein. For example, folding of the model peptide R-helix (4-6) and -hairpin fragment (7) occurs in nanoseconds to a few microseconds. These observations raise the possibility that small single-domain proteins could also have submillisecond folding phases in which secondary structural elements are seeded for facilitat- ing efficient and biased folding. Fast folding experiments have thus attracted the attention of many for over a decade. The first ultrafast experiment was based on the fact that the two-state folding transition (N h U) of ferrocytochrome c (ferrocyt c) is shifted to a lower concentration of guanidine hydrochloride (GdnHCl) 1 when the solvent is saturated with ∼1 mM CO (8, 9). The shift occurs because of preferential binding of CO to the unfolded protein. Within a narrow range of GdnHCl, between ∼4.2 and 4.7 M, the CO-bound protein is unfolded, but the CO-free form is nativelike. It follows that photodissociation of CO within this window of GdnHCl will initiate folding (8). However, this is an extremely poor condition to study protein folding, since the required concentration of the denaturant to make the experiment work is just too close to the transition midpoint (∼5.1 M GdnHCl) of ferrocyt c (9). A similar photochemical protocol that takes advantage of the stability difference of the two oxidation states of cyt c in order to initiate refolding of ferrocyt c by rapidly injecting an electron into the ferric heme of unfolded ferricyt c (10) works only under conditions where no substantial driving force for folding is available. In other approaches, laser T-jump methods have been used to monitor fast folding events for a number of peptides and proteins, including barstar (11), apomyoglobin (12-14), En- HD protein (15), and λ 6-85 protein (16). An elegant applica- tion of laser flash photolysis for direct estimation of the speed limit for protein folding has been described (17). Ultrarapid mixing methods have been introduced to measure the earliest folding events in ferricyt c (18-21), acyl-CoA binding protein (22), the four-helix protein Im7 (23), and -lacto- globulin (24). NMR and hydrogen-exchange methods have been used for microsecond studies of monomeric λ repressor (25) and a crossed-link variant of the GCN4 coiled coil (26). For several reasons, however, carbonmonoxy-cyt c con- tinues to be an indispensable standard system in the fast- folding field. Being a single-domain fast-folding protein, ferrocyt c is paradigmatic (9, 27-30). Since photodissocia- tion of CO occurs in subpicosecond times (31), there is virtually no dead time in probing the dynamics of the refolding polypeptide. The presence of the heme renders possible the use of a variety of additional spectroscopic probes in the time-resolved mode, including optical spec- troscopy and magnetic circular dichroism (32, 33). Even † This work was supported by grants from the Department of Biotechnology (BRB/15/227/2001) and the Department of Science and Technology (4/1/2003-SF) and by the University Grants Commission (UPE Funding), Government of India. A.K.B. is the recipient of a Swarnajayanti Fellowship from the Department of Science and Technology. * To whom correspondence should be addressed. E-mail: akbsc@ uohyd.ernet.in. Phone: 91-40-2313-4810. Fax: 91-40-2301-2460. 1 Abbreviations: GdnHCl, guanidine hydrochloride; cyt c, cyto- chrome c; ferricyt c, ferricytochrome c; ferrocyt c, ferrocytochrome c; carbonmonoxy-cyt c, carbon monoxide-bound ferrocytochrome c; TOF, time of flight; ∆GD, Gibbs energy of denaturation; ∆GD°, Gibbs energy of denaturation in the absence of denaturant. 9359 Biochemistry 2005, 44, 9359-9367 10.1021/bi050384b CCC: $30.25 © 2005 American Chemical Society Published on Web 06/10/2005