Kinetic study of protein unfolding and refolding using urea gradient electrophoresis

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The kinetics of unfolding and refolding of several well-studied proteins have been examined by rapid electrophoresis in polyacrylamide gels containing a linear gradient of urea perpendicular to the direction of migration, comparing the patterns obtained with native and with unfolded proteins. The temperature was 2 °C, so as to decrease the rates of folding transitions; in particular, the half-time for cis-trans isomerization of peptide bonds adjacent to proline residues in fully unfolded proteins should be comparable to, or longer than, the time required for electrophoresis. Slow-refolding forms of the unfolded protein, which probably result from incorrect cis-trans isomers, were demonstrated directly with ribonuclease, chymotrypsinogen and α-chymotrypsin, but they refolded at low urea concentrations more rapidly than would be expected for simple proline cis-trans isomerization. Furthermore, the expected slow-refolding species of lysozyme, α-lactalbumin, staphylococcal nuclease, cytochrome c and β-lactoglobulins were not detected. It is likely that incorrect cis-trans isomers in the unfolded forms of these proteins do not completely block refolding. Where residual unfolded protein was observed transiently in urea concentrations favouring refolding, it invariably tended to form, rapidly and reversibly, labile conformations which were very compact, but not native-like. In contrast, transient folded protein under conditions favouring unfolding did not show any indication of partial unfolding. This indicates that the rate-limiting step in both folding and unfolding generally is very near to the native-like conformation when the reaction co-ordinate of folding is measured by the compactness of the protein molecule. This is inconsistent with at least some popular models of protein folding.