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Structural characterization of an equilibrium unfolding intermediate in cytochrome c.
J Mol Biol. 2006 Mar 31; 357(3):1009-25.JM

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 His-heme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1H-15N 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 alpha-helical sub-domain, 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.

Authors+Show Affiliations

Latypov RF
Basic Science Division, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Cheng H
No affiliation info available
Roder NA
No affiliation info available
Zhang J
No affiliation info available
Roder H
No affiliation info available

MeSH

Amino Acid SubstitutionAnimalsCytochromes cGuanidineHorsesProtein DenaturationProtein FoldingSpectrophotometryUrea

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

16473367

Citation

Latypov, Ramil F., et al. "Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome C." Journal of Molecular Biology, vol. 357, no. 3, 2006, pp. 1009-25.
Latypov RF, Cheng H, Roder NA, et al. Structural characterization of an equilibrium unfolding intermediate in cytochrome c. J Mol Biol. 2006;357(3):1009-25.
Latypov, R. F., Cheng, H., Roder, N. A., Zhang, J., & Roder, H. (2006). Structural characterization of an equilibrium unfolding intermediate in cytochrome c. Journal of Molecular Biology, 357(3), 1009-25.
Latypov RF, et al. Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome C. J Mol Biol. 2006 Mar 31;357(3):1009-25. PubMed PMID: 16473367.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural characterization of an equilibrium unfolding intermediate in cytochrome c. AU - Latypov,Ramil F, AU - Cheng,Hong, AU - Roder,Navid A, AU - Zhang,Jiaru, AU - Roder,Heinrich, Y1 - 2006/02/03/ PY - 2005/11/11/received PY - 2006/01/11/revised PY - 2006/01/15/accepted PY - 2006/2/14/pubmed PY - 2006/5/11/medline PY - 2006/2/14/entrez SP - 1009 EP - 25 JF - Journal of molecular biology JO - J Mol Biol VL - 357 IS - 3 N2 - 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 His-heme ligation. For a more detailed structural characterization of the intermediate, we used 2D 1H-15N 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 alpha-helical sub-domain, 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. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/16473367/Structural_characterization_of_an_equilibrium_unfolding_intermediate_in_cytochrome_c_ DB - PRIME DP - Unbound Medicine ER -