ABSTRACT
The change in hydrodynamic volume accompanying the reversible folding of a protein into its native structure can be easily observed by high-performance size-exclusion chromatography (SEC) as a change in the elution time of the protein. Three kinds of profiles are necessary for a complete analysis. Folding profiles are obtained by injection of an unfolded protein in a SEC column equilibrated with buffer or a low concentration of a protein denaturant. Unfolding profiles are obtained by injection of a native protein in a column equilibrated with a high concentration of denaturant. Equilibrium profiles are obtained by equilibration of both the protein and the column with the same concentration of denaturant prior to injection. Inspection of such profiles can identify the participation of intermediate structures in the folding process while analysis by computer simulation can establish the refolding mechanism and the equilibrium and kinetic parameters of each component in the mechanism.
