Ultrafast Events in the Folding of Ferrocytochrome c

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 degrees C. Under the absolute refolding condition, the earliest relaxation time of the unfolded protein chain is less than 1 micros. 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 approximately 0.4 and approximately 3 micros. 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 approximately 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.

Analysis of the pH-dependent stability and millisecond folding kinetics of horse cytochrome c

“…Similarly, the fit of data in Fig. 2 This result is consistent with an earlier report which showed that denaturantinduced unfolding of Ferrocyt c occurs in a two-state manner [70].…”

“…Fig. 5 C presents the GdnHCl-dependence of the logarithm of observed refolding and unfolding rates of Ferrocyt c measured at pH 7.0 and pH 12.7 at 25 C. Our previous extensive equilibrium and millisecond kinetic folding studies of Ferrocyt c revealed that the folding kinetics of Ferrocyt c is two-state [70], so in the current manuscript the dependency of Log k obs on GdnHCl concentration was analyzed according to two-state model, N#U [52],…”

“…In stopped-flow experiments, GdnHCl-unfolded Ferrocyt c (Ustate) in native buffer at pH 7.0 refolds rapidly to the native-state (N-state) [70,71]. Fig.…”

“…This finding demonstrates that the large disparity in thermodynamic stability of protein at pH 12.7 relative to pH 7.0 is not greatly reflected in the refolding rates, which could be due to screened chargeecharge interactions in the presence of 1 M GdnHCl. To test this aspect further, similar kind of experiments need to be performed using nonionic denaturant such as urea but within the limit of aqueous solubility of urea Ferrocyt c exhibits incomplete unfolding [70,90]. Fig.…”

Sub-Second Cellular Dynamics: Time-Resolved Electron Microscopy and Functional Correlation

“…This tech nique serves preferably to determine enzyme kinetics (Cherepanov and DeVries, 2004;Gutfreund. 1999;Purich, 2002), protein folding (Kumar et al. 2005), metabolite formation (Genazzani et al, 1997), ligand-receptor interactions (Hess, 1993: Sklar et a/.…”