Folding kinetics of a fluorescent variant of monomeric λ repressor

A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degrees C using fluorescence stopped-flow and dynamic NMR. Equilibrium denaturation curves obtained by circular dichroism, fluorescence, and NMR are superimposable. Stopped-flow analysis indicates that in the absence of denaturants the folding reaction is complete within the dead-time of the experiment. Within higher denaturant conditions, where the folding rate is slower, NMR and stopped-flow agree on the folding and unfolding rates of the protein. In 3.4 M urea and 1.8 M GdmCl, we show that the variant folds within 2 ms. Extrapolation indicates that the folding time is 20 mu s in the absence of denaturants, All folding and unfolding reactions displayed monoexponential kinetics, and no burst-phases were observed. In addition, the thermodynamic parameters Delta G and m(eq) obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state D<->N folding model.