Major differences in stability and dimerization properties of two chimericmutants of human stefins

Stefins A and B are cysteine proteinase inhibitors that have considerable s equence similarity but marked differences in their stability and folding pr operties. Two chimeric proteins were designed to shed light on these differ ences. The chimeric mutants have been expressed in Escherichia coli and hav e been isolated. The first, A37B, consists of 37 residues of stefin A, comp rising 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, follo wed by 37 residues of stefin B. Spectroscopic properties of the chimeric pr oteins (absorption, CD, and NMR spectra), together with activity measuremen ts, have confirmed that both have well-defined tertiary structure and are a ctive as cysteine proteinase inhibitors. Characterization consisted of GuHC l denaturation, ANS binding as a function of pH, and monitoring of dimeriza tion 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 A3 7B as compared with 1.4 M GuHCl for stefin B tall at pH 7.5, 25 degreesC). However (G degrees (N-U) is lower for both chimeric proteins (18 +/- 3 kJ/m ol) than for the parent stefins (28 +/- 3 kJ/mol), In pH denaturation, unli ke 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 t hey remain "trapped" only in stefin A. (C) 2001 Wiley-Liss, Inc.