A tryptophan-containing variant of monomeric lambda repressor has been made, and its folding kinetics were analyzed at 20 degreesC 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 micro(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 meq obtained from the kinetic analysis are consistent with the equilibrium experiments. The results support a two-state Dleft and right arrow N folding model.
Arc repressor bearing the N11L substitution (Arc-N11L) is an evolutionary intermediate between the wild type protein, in which the region surrounding position 11 forms a beta-sheet, and a double mutant 'switch Arc', in which this region is helical. Here, Arc-N11L is shown to be able to adopt either the wild type or mutant conformations. Exchange between these structures occurs on the millisecond time scale in a dynamic equilibrium in which the relative populations of each fold depend on temperature, solvent conditions and ligand binding. The N11L mutation serves as an evolutionary bridge from the beta-sheet to the helical fold because in the mutant, Leu is an integral part of the hydrophobic core of the new structure but can also occupy a surface position in the wild type structure. Conversely, the polar Asn 11 side chain serves as a negative design element in wild type Arc because it cannot be incorporated into the core of the mutant fold.
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