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Stefins A and B are cysteine proteinase inhibitors that have considerable sequence similarity but marked differences in their stability and folding properties. Two chimeric proteins were designed to shed light on these differences. The chimeric mutants have been expressed in Escherichia coli and have been isolated. The first, A37B, consists of 37 residues of stefin A, comprising the N-terminal and the alpha-helix, joined to 61 residues of stefin B; the second, A61B, consists of 61 N-terminal residues of stefin A, followed by 37 residues of stefin B. Spectroscopic properties of the chimeric proteins (absorption, CD, and NMR spectra), together with activity measurements, have confirmed that both have well-defined tertiary structure and are active as cysteine proteinase inhibitors. Characterization consisted of GuHCl denaturation, ANS binding as a function of pH, and monitoring of dimerization under partially denaturing conditions. The c(m) values are 1.3 M GuHCl for A61B as compared with 2.7 M GuHCl for stefin A, and 2.1 M GuHCl for A37B as compared with 1.4 M GuHCl for stefin B (all at pH 7.5, 25 degrees C). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/mol) than for the parent stefins (28 +/- 3 kJ/mol). In pH denaturation, unlike stefin B, neither chimeric mutant unfolds to I(N) below pH 5.4. At pH 3, where stefin B forms a molten globule and stefin A is native, both A37B and A61B show increased ANS fluorescence and aggregate visibly. Dimers at pre-denaturation conditions are observed in all the proteins under study, but they remain "trapped" only in stefin A.

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.