Protein denaturation:: A small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c

被引:129
作者
Segel, DJ
Fink, AL
Hodgson, KO
Doniach, S
机构
[1] Stanford Univ, Dept Phys, Stanford, CA 94305 USA
[2] Stanford Univ, Dept Chem, Stanford, CA 94305 USA
[3] Univ Calif Santa Cruz, Dept Chem & Biochem, Santa Cruz, CA 95064 USA
[4] Stanford Synchrotron Radiat Lab, Stanford, CA 94309 USA
关键词
D O I
10.1021/bi980535t
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Solution X-ray scattering was used to study the equilibrium unfolding of cytochrome c as a function of guanidine hydrochloride concentration at neutral pH. The radius of gyration (R-g) shows a cooperative transition with increasing denaturant with a similar C-m to that observed with circular dichroism. However, the lack of an isoscattering point in the X-ray scattering patterns suggests the equilibrium unfolding is not simply a two-state process. Singular value decomposition (SVD) analysis was applied to the scattering patterns to determine the number of distinct scattering species. SVD analysis reveals the existence of three components, suggesting that at least three equilibrium states of the protein exist. A model was employed to determine the thermodynamic parameters and the scattering profiles of the three equilibrium states. These scattering profiles show that one state is native (N). The other two states (U-1, U-2) are unfolded, with U-2 being fully unfolded and U-1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U-1 is maximally populated at intermediate denaturant concentrations while U-2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U-1 and U-2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.
引用
收藏
页码:12443 / 12451
页数:9
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