Kenig M, Jerala R, Kroon-Zitko L, Turk V, Zerovnik E
Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
Proteins. 2001 Mar 1;42(4):512-22.
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.
丝氨酸蛋白酶抑制剂A和B是半胱氨酸蛋白酶抑制剂，它们具有相当的序列相似性，但在稳定性和折叠特性上有显著差异。设计了两种嵌合蛋白来阐明这些差异。嵌合突变体已在大肠杆菌中表达并分离出来。第一种，A37B，由丝氨酸蛋白酶抑制剂A的37个残基组成，包括N端和α螺旋，与丝氨酸蛋白酶抑制剂B的61个残基相连；第二种，A61B，由丝氨酸蛋白酶抑制剂A的61个N端残基组成，接着是丝氨酸蛋白酶抑制剂B的37个残基。嵌合蛋白的光谱性质（吸收光谱、圆二色光谱和核磁共振光谱）以及活性测量结果证实，两者都具有明确的三级结构，并且作为半胱氨酸蛋白酶抑制剂具有活性。表征包括盐酸胍变性、ANS结合随pH值的变化以及在部分变性条件下二聚化的监测。A61B的c(m)值为1.3 M盐酸胍，而丝氨酸蛋白酶抑制剂A为2.7 M盐酸胍；A37B为2.1 M盐酸胍，而丝氨酸蛋白酶抑制剂B为1.4 M盐酸胍（均在pH 7.5、25℃下）。然而，两种嵌合蛋白的(G°(N-U)均低于亲本丝氨酸蛋白酶抑制剂（分别为18±3 kJ/mol和28±3 kJ/mol）。在pH变性过程中，与丝氨酸蛋白酶抑制剂B不同，两种嵌合突变体在pH 5.4以下均不会展开为I(N)。在pH 3时，丝氨酸蛋白酶抑制剂B形成熔球态，丝氨酸蛋白酶抑制剂A为天然态，A37B和A61B均显示出ANS荧光增强并明显聚集。在所有研究的蛋白质中，在预变性条件下均观察到二聚体，但只有在丝氨酸蛋白酶抑制剂A中它们会保持“被困”状态。
