Distinguishing between two-state and three-state models for ubiquitin folding

Conflicting results exist regarding whether the folding of mammalian ubiqui tin at 25 degrees C is a simple, two-state kinetic process or a more comple x, three-state process with a defined kinetic intermediate. We have measure d folding rate constants up to about 1000 s(-1) using conventional rapid mi xing methods in single-jump, double-jump, and continuous-flow modes. The li near dependence of folding rates on denaturant concentration and the lack o f an unaccounted "burst-phase" change for the fluorescence signal indicate that a two-state folding model is adequate to describe the folding pathway. This behavior also is seen for folding in the presence of the stabilizing additives 0.23 M sodium sulfate and 1 M sodium chloride. These results stre ss the need for caution in interpreting deviations from ideal two-state "ch evron" behavior when folding is heterogeneous or folding rate constants are near the detection limit.