Equilibrium and kinetic measurements of the conformational transition of thioredoxin in urea

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Abstract
Addition of urea to solutions of Escherichia coli thioredoxin results in a cooperative unfolding of the protein centered at 6.7 M urea at 25.degree. C and 5.1 M urea at 2.degree. C and neutral pH as judged by changes in tryptophan fluorescence emission, far-ultraviolet circular dichroism, and exclusion chromatography. Kinetic profiles of changes in tryptophan fluorescenc emission intensity were analyzed following either manual or stopped-flow mixing to initiate unfolding or refolding. Unfolding of the native protein occurs in a single kinetic phase whose time constant is markedly dependent on urea concentration. Refolding of the urea-denatured protein occurs in a multiplicity of kinetic phases whose time constants and fractional amplitudes are also dependent upon urea concentration. Urea gradient gel electrophoresis and exclusion chromatographic measurements suggest the transient accumulation of at least one and likely two compact nativelike intermediate conformations during refolding. Simulations of both electrophoretic and chromatographic results suggest that the intermediate conformations are generated by the concerted action of the middle and fast refolding phases.