On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states

It has been shown that hu- man stefin B exhibits molten globule intermedi- ates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296- 303), the kinetics measured by circular dichro- ism (CD) and fluorescence were correlated. In the present work the kinetics of folding were moni- tored by tyrosine fluorescence, ANS fluores- cence, and, for certain reactions, far ultravio- let (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid dena- tured state at pH 1.8 6 0.2, an acid molten globule intermediate I1 (pH 3.3 6 0.1, low salt), a more structured acid molten globule interme- diate I2 (pH 3.3 6 0.1, 0.42 M NaCl), and the TFE state (pH 3.3 6 0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identi- cal. This does not apply to the second acid molten globule intermediate I2, which demon- strates a higher rate of folding by a factor of 270. Different energy of activation and pH depen- dence were found for folding from states I1 or I2. Proteins 32:304-313, 1998. r 1998 Wiley-Liss, Inc.