Early events, kinetic intermediates and the mechanism of protein folding in cytochrome C

Abstract

Kinetic studies of the early events in cytochrome c folding are reviewed with a focus on the evidence for folding intermediates on the submillisecond timescale. Evidence from time-resolved absorption, circular dichroism, magnetic circular dichroism, fluorescence energy and electron transfer, small-angle X-ray scattering and amide hydrogen exchange studies on the t < or = 1 ms timescale reveals a picture of cytochrome c folding that starts with the approximately 1-micros conformational diffusion dynamics of the unfolded chains. A fractional population of the unfolded chains collapses on the 1 - 100 micros timescale to a compact intermediate I(C) containing some native-like secondary structure. Although the existence and nature of I(C) as a discrete folding intermediate remains controversial, there is extensive high time-resolution kinetic evidence for the rapid formation of I(C) as a true intermediate, i.e., a metastable state separated from the unfolded state by a discrete free energy barrier. Final folding to the native state takes place on millisecond and longer timescales, depending on the presence of kinetic traps such as heme misligation and proline mis-isomerization. The high folding rates observed in equilibrium molten globule models suggest that I(C) may be a productive folding intermediate. Whether it is an obligatory step on the pathway to the high free energy barrier associated with millisecond timescale folding to the native state, however, remains to be determined.

Keywords: Collapsed intermediate; Trp59 fluorescence; amide hydrogen exchange; conformational diffusion; disordered tertiary structure; far-UV circular dichroism; heme misligation; magnetic circular dichroism; molten globule; secondary structure formation; small-angle X-ray scattering; thermophiles; three-state pathway; time-resolved spectroscopy; unfolded chains.

Figure 1.

Drawing of the native structure of horse heart cytochrome c showing the heme coordinated by His18 and Met80, the locations of His26 and His33 (which can bind to heme under denaturing conditions), and Trp59 (PDB 1hrc). The foldons, discrete units of secondary and tertiary structure that unfold sequentially in the order red, yellow, green, and blue, are shown as identified by Bai et al. [16] (see text).

Figure 2.

Characteristic timescales of dynamical processes during the folding of cytochrome c.

Figure 3.

Equilibrium populations of native (N), molten globule (M), and unfolded (U) states of Fe^II (blue lines) and Fe^III (red lines) cyt c vs. GuHCl concentration, calculated from the thermodynamic data of Thomas et al., pH 6.5, 40 °C [24].

Figure 4.

Schematic of a possible cytochrome c folding pathway U ⇔ I_C → N shown as free energy vs. reaction coordinate. The equilibrium observed for the first step implies that I_C lies within ~ k_BT in free energy from U. Formation of the (Blue) N-/C-terminal helix foldon is the rate-limiting barrier to folding (TS). After passage over TS, the remaining foldons (Green, Yellow, and Red) are formed very rapidly through a sequence of ephemeral intermediates leading to N. (For simplicity, the foldon labels are shown by the respective energy barriers leading to their formation.).