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Latypov Ramil F , Cheng Hong , Roder Navid A , Zhang Jiaru , Roder Heinrich
Structural Characterization of an Equilibrium Unfolding Intermediate in Cytochrome c
Journal of Molecular Biology. 2006 ;357(3) :1009-1025
PMID: AN 2006:258143   
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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 equil. intermediate. However, little is known about the structural and thermodn. properties of this state, and whether it is stabilized by the presence of non-native heme ligands. We monitored the reversible denaturant-induced unfolding equil. 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 quant. interpretation of the results in terms of a three-state model, which enabled us to det. the intrinsic spectroscopic properties of the intermediate. A well-populated intermediate was obsd. in equil. expts. 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 equil. state obsd. in our optical and NMR studies contains many native-like structural features, including a well-structured a-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 alk. 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. [on SciFinder (R)]
6 General Biochemistry Basic Science Division,Fox Chase Cancer Center, 333 Cottman Avenue,Philadelphia,PA,USA. Journal 0022-2836 written in English.