Structural characterization of an equilibrium unfolding intermediate in cytochrome c.


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. Study holds ProTherm entries: 22142, 22143, 22144, 22145, 22146, 22147, 22148, 22149, 22150 Extra Details: native to intermediate; thermodynamic parameters by global fitting of a three state model to the GdnHCl-induced unfolding transitions monitored by heme absorbance, near and far-UV CD and fluorescence. protein folding; denaturation; stability; spectroscopy; NMR

Submission Details

ID: MkdYZbk9

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:53 p.m.

Version: 1

Publication Details
Latypov RF;Cheng H;Roder NA;Zhang J;Roder H,J. Mol. Biol. (2006) Structural characterization of an equilibrium unfolding intermediate in cytochrome c. PMID:16473367
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Cytochrome c P00004 CYC_HORSE
99.0 Cytochrome c P68097 CYC_EQUAS
99.0 Cytochrome c P68096 CYC_EQUBU
97.1 Cytochrome c P62894 CYC_BOVIN
97.1 Cytochrome c P62895 CYC_PIG
97.1 Cytochrome c P62896 CYC_SHEEP
94.3 Cytochrome c P00007 CYC_HIPAM
95.2 Cytochrome c P68099 CYC_CAMDR
95.2 Cytochrome c P68100 CYC_ESCRO
95.2 Cytochrome c P68098 CYC_LAMGU
94.3 Cytochrome c P62897 CYC_MOUSE
94.3 Cytochrome c P00008 CYC_RABIT
94.3 Cytochrome c P62898 CYC_RAT
94.3 Cytochrome c P00011 CYC_CANLF
93.3 Cytochrome c P00014 CYC_MACGI
93.3 Cytochrome c P00013 CYC_MINSC
93.3 Cytochrome c Q52V09 CYC_CEPBA
93.3 Cytochrome c P00012 CYC_MIRLE
90.5 Cytochrome c Q52V10 CYC_SAISC
92.3 Cytochrome c P81280 CYC_ALLMI
92.2 Cytochrome c P00020 CYC_ANAPL
91.3 Cytochrome c P00021 CYC_COLLI
90.3 Cytochrome c B4USV4 CYC_OTOGA