Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c

Abstract

Although the denaturant-induced unfolding transition of cytochrome c was initially thought to be a cooperative process, recent spectroscopic studies have shown deviations from two-state behavior consistent with accumulation of an equilibrium intermediate. However, little is known about the structural and thermodynamic properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equilibrium of oxidized horse cytochrome c using various spectroscopic probes, including fluorescence, near and far-UV CD, heme absorbance bands in the Soret, visible and near-IR regions of the spectrum, as well as 2D NMR. Global fitting techniques were used for a quantitative interpretation of the results in terms of a three-state model, which enabled us to determine the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was observed in equilibrium experiments at pH 5 using either guanidine-HCl or urea as a denaturant, both for wild-type cytochrome c as well as an H33N mutant chosen to prevent formation of non-native Hisheme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1 H-15 N correlation spectroscopy to follow the changes in peak intensity for individual backbone amide groups. The equilibrium state observed in our optical and NMR studies contains many native-like structural features, including a well-structured a-helical subdomain, a short Trp59-heme distance and solvent-shielded heme environment, but lacks the native Met80 sulfur-iron linkage and shows major perturbations in side-chain packing and other tertiary interactions. These structural properties are reminiscent of the A-state of cytochrome c, a compact denatured form found under acidic high-salt conditions, as well as a kinetic intermediate populated at a late stage of folding. The denaturant-induced intermediate also resembles alkaline forms of cytochrome c with altered heme ligation, suggesting that disruption of the native methionine ligand favors accumulation of structurally analogous states both in the presence and absence of non-native ligands.

Zinc porphyrin: A fluorescent acceptor in studies of Zn-cytochromecunfolding by fluorescence resonance energy transfer

“…This may reflect an expansion of the protein without loss of helical content before the transition from the folded state to the intermediate state. This expansion at low denaturant concentrations has not been reported in previous studies of cyt c denaturation using CD, absorption, or fluorescence (19,28,29). The existence of an expansion is consistent with the observation that low concentrations of GuHCl increase hydrogen-deuterium exchange rates in cyt c as higher-energy nonnative conformations become populated (30).…”

“…This model was chosen because a number of studies of cyt c folding have successfully used a three-state model for interpretation of results (28,31,32). In the three-state model used, the protein unfolds from the native state (N) through one intermediate (I) to the unfolded state (U).…”

“…Here, the distance between AF and ZnP in the intermediate I for each variant provides insight into the structure of the intermediate. For example, analysis of NMR, CD, and absorption data suggests that I retains overall native-like structure while the protein ''loosens'' up (28), and another NMR analysis has shown that the N-to-I transition has a small overall m 1 value (31). Our results are consistent with this analysis: the AF-ZnP distance changes for almost all AF label positions for the transition from N to I are small in comparison with the distance changes observed in the I-to-U transition (position 4 is the exception, discussed subsequently).…”

“…Notably, residue 4 is only 10 residues from Cys-14, to which ZnP is covalently attached, thus limiting the AF-ZnP distance change for this position. Position 99 has a relatively large m 1 It is informative to compare these results with an NMR study of cyt c denaturation, whereby persistence of a native-like chemical environment for residues on helices 1 and 4 (Asp-2 and Asn-103) in increasing denaturant was observed, relative to probe nuclei on loop 1 (Gly-37), helix 2 (Thr-49), and loop 3 (Met-80), which show changes in local structure at low denaturant concentrations (28). This may seem contradictory to our result that helix 1, as probed at position 4, experiences its more significant unfolding event in the transition from N to I.…”

Investigation on the Conformation Change of Hemoglobin Immobilized on MPA-modified Electrode by Electrochemical Method

“…24 Three-, four-and five-state unfolding processes of some proteins by different denaturants have been reported by some researchers. [25][26][27][28][29] These works not only need fluorescence spectroscopy, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, infrared spectroscopy, size exclusion chromatography and other physical and chemical means, but require complex testing and complicated calculations. However, to our knowledge, there have been no reports on the stable conformational state distribution during the protein unfolding process by electrochemical method.…”