Major differences in stability and dimerization properties of two chimeric mutants of human stefins.
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.
Kenig
M
M
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Jerala
R
R
Kroon-Zitko
L
L
Turk
V
V
Zerovnik
E
E
D002942:Circular Dichroism; D055312:Cystatin A; D055313:Cystatin B; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004795:Enzyme Stability; D005453:Fluorescence; D019791:Guanidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
C524994:CSTB protein, human; D055312:Cystatin A; D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C026407:CSTA protein, human; D055313:Cystatin B; D019791:Guanidine