THE FOLDING MECHANISM OF BARSTAR - EVIDENCE FOR MULTIPLE PATHWAYS ANDMULTIPLE INTERMEDIATES

The mechanism of folding of the small protein barstar in the pre-trans ition zone at pH 7, 25 degrees C has been characterized using rapid mi xing techniques. Earlier studies had established the validity of the t hree-state U-S reversible arrow U-F reversible arrow N mechanism for f olding and unfolding in the presence of guanidine hydrochloride (GdnHC l) at concentrations greater than 2.0 M, where U-S and U-F are the slo w-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-S2 as well as a late native-like intermediate, I-N, are present on the folding pathways of U-S, and an early intermediate I-F1 on the folding pathway of U-F, when bars tar 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 G dnHCl in the pre-transition zone have been measured. The data indicate that in 1.5 M GdnHCl, I-N is not fully populated on the U-S --> I-S1 --> I-N --> N pathway because the rate of its formation is so slow tha t the U-S reversible arrow U-F reversible arrow N pathway can effectiv ely compete with that pathway. In 1.0 M GdnHCl, the U-S --> I-S1 --> I -N transition 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 intermedi ates, I-M1 and I-M2, that form within 2 ms on the U-S --> I-M1 --> I-S 1 --> 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 th e folding pathway of U-F.