title: Protein denaturation: a small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c.
abstract: 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 (Rg) shows a cooperative transition with increasing denaturant with a similar Cm 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 (U1, U2) are unfolded, with U2 being fully unfolded and U1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U1 is maximally populated at intermediate denaturant concentrations while U2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U1 and U2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.
authors.affiliation: Department of Physics, Stanford University, California 94305, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Segel
authors.forename: A L
authors.initials: AL
authors.lastname: Fink
authors.forename: K O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: S
authors.initials: S
authors.lastname: Doniach
mesh_terms: D000818:Animals; D003574:Cytochrome c Group / Q000737:chemistry*; D019791:Guanidine; D006736:Horses; D008956:Models, Chemical; D011489:Protein Denaturation; D017510:Protein Folding; D012542:Scattering, Radiation; D013816:Thermodynamics; D014965:X-Rays
chemical_list: D003574:Cytochrome c Group; D019791:Guanidine