FOLDING KINETICS OF A FLUORESCENT VARIANT OF MONOMERIC LAMBDA-REPRESSOR

A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degrees C using fl uorescence stopped-flow and dynamic NMR. Equilibrium denaturation curv es obtained by circular dichroism, fluorescence, and NMR are superimpo sable. Stopped-flow analysis indicates that in the absence of denatura nts the folding reaction is complete within the dead-time of the exper iment. 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 varian t folds within 2 ms. Extrapolation indicates that the folding time is 20 mu s in the absence of denaturants, All folding and unfolding react ions displayed monoexponential kinetics, and no burst-phases were obse rved. In addition, the thermodynamic parameters Delta G and m(eq) obta ined from the kinetic analysis are consistent with the equilibrium exp eriments. The results support a two-state D<->N folding model.