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The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25 degrees C has been characterized using rapid-mixing techniques. Earlier studies had established the validity of the three-state US <--> UF <--> N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where US and UF are the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, IS1 and IS2 as well as a late native-like intermediate, IN, are present on the folding pathways of US, and an early intermediate IF1 on the folding pathway of UF, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of IN, and the rates of formation of N at three different concentrations of GdnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, IN is not fully populated on the US-->IS1-->IN-->N pathway because the rate of its formation is so slow that the US <--> UF <--> N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the US-->IS1-->IN transition is so fast that IN is fully populated. In 0.6 M GdnHCl, IN appears not to be fully populated because an alternative folding pathway, US-->IS2-->N, becomes available for the folding of US, in addition to the US-->IS1-->IN-->N pathway. Measurement of the binding of the hydrophobic dye 1-anilino-8-naphthalenesulphonate (ANS) during folding indicates that ANS binds to two distinct intermediates, IM1 and IM2, that form within 2 ms on the US-->IM1-->IS1-->IN-->N and US-->IM2-->IS2-->N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of UF.

The fluorescence-monitored kinetics of folding and unfolding of barstar by guanidine hydrochloride (GdnHCl) in the folding transition zone, at pH 7, 25 degrees C, have been quantitatively analyzed using a 3-state mechanism: U(S)<-->UF<-->N. U(S) and UF are slow-refolding and fast-refolding unfolded forms of barstar, and N is the native protein. U(S) and UF probably differ in possessing trans and cis conformations, respectively, of the Tyr 47-Pro 48 bond. The 3-state model could be used because the kinetics of folding and unfolding of barstar show 2 phases, a fast phase and a slow phase, and because the relative amplitudes of the 2 phases depend only on the final refolding conditions and not on the initial conditions. Analysis of the observed kinetics according to the 3-state model yields the values of the 4 microscopic rate constants that describe the transitions between the 3 states at different concentrations of GdnHCl. The value of the equilibrium unfolded ratio U(S):UF (K21) and the values of the rate constants of the U(S)-->UF and UF-->U(S) reactions, k12 and k21, respectively, are shown to be independent of the concentration of GdnHCl. K21 has a value of 2.1 +/- 0.1, and k12 and k21 have values of 5.3 x 10(-3) s-1 and 11.2 x 10(-3) s-1, respectively. Double-jump experiments that monitor reactions that are silent to fluorescence monitoring were used to confirm the values of K21, k12, and k21 obtained from the 3-state analysis and thereby the validity of the 3-state model.(ABSTRACT TRUNCATED AT 250 WORDS)