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Correspondence between anomalous m- and C_p-values in protein folding

¹ Department of Life Sciences, Aalborg University, DK-9000 Aalborg, Denmark
² Department of Biochemistry, Umeå University, S-90187 Umeå, Sweden

(RECEIVED July 14, 2004; FINAL REVISION August 24, 2004; ACCEPTED August 26, 2004)

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10°C to 70°C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant–protein interactions is a major determinant of the response of energy landscape contours to changing environments.

Keywords: protein folding; kinetics; thermodynamics; transition state ensemble; m-values; heat capacity

Abbreviations: C_p^D–N, heat capacity difference between the native and denatured state • C_p^f, activation heat capacity of folding • C_p^u, activation heat capacity of unfolding • TSE, transition state ensemble

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04991004.

Reprint requests to: Daniel E. Otzen, Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark; e-mail: dao{at}bio.aau.dk; fax: +45-98-14-18-08.

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