Protein Folding in Classical Perspective:  Folding of Horse Cytochrome c

Abstract

Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), respectively. But the large disparity in thermodynamic stability is not strongly reflected in their refolding rates. Cross-pH studies, where GdnHCl-unfolded states of neutral- and alkaline ferrocyt c are allowed to refold to the same final pH and denaturant concentration, indicate that the refolding rates are largely independent of the stability, configuration, ionization, and solvation of the initial unfolded state. Also, burst relaxation signals in cross-pH refolding runs show the same quantitative dependence on GdnHCl, suggesting that the earliest relaxation or reconfiguration of the chains must be the same and is independent of the initial equilibrium unfolded state. Analyses along the classical line indicate an early transition state where much less than a third of the protein surface that is buried in the native state becomes buried. The barrier energy is of the order of 10 k(B)T. The results, apparently inconsistent with the predictions of the funnel model, afford a mechanistic description of folding in which the folding time of small single-domain proteins is set by the time needed for the denatured polypeptide to search-find a nativelike topology.

Folding Mechanism of Reduced Cytochrome c: Equilibrium and Kinetic Properties in the Presence of Carbon Monoxide

“…Global fitting of a two-state model to the optically monitored unfolding transitions of Fe 2+ cyt c at 20 °C (in 0.1 M sodium phosphate, pH 7.0) yields an average midpoint C m = 5.22 ± 0.06 M and m -value m NU = 3.25 ± 0.24 kcal mol −1 M −1 , corresponding to a free energy ΔG NU = 17.0 ± 1.3 kcal mol −1 . Similar unfolding free energies (18.0–19.3 kcal mol −1 ) have been reported previously for reduced cyt c under similar conditions 49,50…”

“…refs. 49,50) range from 17.0 to 19.3 kcal mol −1 . This discrepancy is related in part to the unusually high midpoint for the GuHCl-induced unfolding transition of Fe 2+ cyt c (5.2 M), which makes it difficult to define the post-transition baseline and introduces considerable uncertainty in ΔG due to the long extrapolation, making the analysis highly sensitive to errors in the m-value.…”

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

“…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.…”

Protein Folding Dynamics of Cytochrome c Seen by Transient Grating and Transient Absorption Spectroscopies

“…[2][3][4][5][6][7][8][9][10][11][12][13][14] In particular, time-resolved spectroscopic techniques with high sensitivity and superb time resolution have been effective in tracking protein folding processes occurring on time scales from nanoseconds to seconds. For example, time-resolved circular dichroism (CD), [3][4][5] IR and Raman, [6][7][8]15 fast mixing stoppedflow method, 16,17 transient absorption (TA) spectroscopy, [16][17][18][19] and laser-induced transient grating (TG) have been applied to studying the dynamics of protein conformational changes. 1,14,[20][21][22] Among these spectroscopic techniques, the TG method is a unique tool that can sensitively probe the global structural changes by monitoring the change of the diffusion coefficient of a protein.…”