Folding kinetics of phage 434 Cro protein

Folding kinetics for phage 434 Cro protein are examined and compared with t hose reported for lambda (6-85), the N-terminal domain of the repressor of phage lambda: The two proteins have similar all-helical structures consisti ng of five helices but different stabilities. In contrast to lambda (6-85), sharp and distinct aromatic H-1 NMR signals without exchange broadening ch aracterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degreesC, indicating slow interconversion on the NMR time scale. Stopped-f low fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfold ing rates, suggesting a native-like transition state ensemble. Refolding ra tes are slower and unfolding rates considerably faster at pH 4 than at pH 6 . This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlin ear urea dependencies that are apparent at pH 6 indicate deviation from two -state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slo wer than <lambda>(6-85) are rationalized in terms of the lower intrinsic he lix stabilities and putative charge interactions in 434 Cro.