Abstract

The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule. Study holds ProTherm entries: 23841, 23842 Extra Details: deltaGU = deltaGU - m[GdnHCl] where deltaGU is the change in free energy of state U relative to the native state at neutral pH and in the absence of denaturants and m is the linear dependence of deltaGU upon the denaturant concentration. Without Imidazole, SAXS= small-angle X-ray scattering; unfolding/refolding kinetics, small-angle x-ray scattering, cytochome c, singular value decomposition.

Submission Details

ID: esYcwWUT3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

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