The Folding mechanism of barstar: evidence for multiple pathways and multiple intermediates

Abstract

The mechanism of folding of the small protein barstar in the pre-transition zone at pH 7, 25°C has been characterized using rapid-mixing techniques. Ear;lier studies had established the validity of the three-state U_S⇌U_F⇌N mechanism for folding and unfolding in the presence of guanidine hydrochloride (GdnHCl) at concentrations greater than 2.0 M, where U_Sand U_F are the slow-refolding and fast-refolding unfolded forms, respectively, and N is the fully folded form. It is now shown that early intermediates, I_S1 and I_S2as well as a late native-like intermediate, I_N, are present on the folding pathways of U_S, and an early intermediate I_F1on folding pathway of U_F, when barstar is refolded in concentrations of GdnHCl below 2.0 M. The rates of formation and disappearance of I_N, 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, I_Nis not fully populated on the U_S→I_S1→I_N→N pathway because the rate of its formation is so slow that the U_S⇌ U_F⇌N pathway can effectively compete with that pathway. In 1.0 M GdnHCl, the U_S→I_S1→I_Ntransition is so fast that I_N is fully populated. In 0.6 M GdnHCl, I_N appears not to be fully populated because an alternative folding pathway, U_S→I_S2→N, becomes available for the folding of U_S, in addition to the U_S→I_S1→I_N→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, I_M1and I_M2, that form within 2 ms on the U_S→I_M1→I_S1→I_N→N and U_S→I_M2→I_S2→N pathways. There is no evidence for the accumulation of intermediates that can bind ANS on the folding pathway of U_F.