Shortle D
J Cell Biochem. 1986;30(4):281-9. doi: 10.1002/jcb.240300402.
Several mutant forms of staphylococcal nuclease with one or two defined amino acid substitutions have been purified, and the effects of the altered amino acid sequence on the stability of the folded conformation have been analyzed by guanidine hydrochloride denaturation. Two nuc- mutations, which greatly reduced the level of enzyme activity accumulated in E coli colonies carrying a recombinant plasmid with the mutant nuc gene (ie, a NUC- phenotype), both result in protein unfolding at significantly lower guanidine hydrochloride concentrations than the wild-type protein, whereas three sup mutations isolated on the basis of their ability to suppress partially the NUC- phenotype of the above two mutations result in unfolding at significantly higher guanidine hydrochloride concentrations. Characterization of nuclease molecules with two different amino acid substitutions, either nuc- + sup pairs or sup + sup pairs, suggests that the effect of an amino acid substitution on the stability of the native conformation, as measured by the value of delta delta GD, may not be a constant, but rather a variable that is sensitive to the presence of other substitutions at distant sites in the same molecule. Surprisingly, the slopes of the log Kapp vs guanidine hydrochloride concentration plots vary by as much as 35% among the different proteins.
已经纯化了几种具有一个或两个特定氨基酸取代的葡萄球菌核酸酶突变形式，并通过盐酸胍变性分析了氨基酸序列改变对折叠构象稳定性的影响。两个核酸酶突变（nuc-）极大地降低了携带突变核酸酶基因的重组质粒的大肠杆菌菌落中积累的酶活性水平（即NUC-表型），这两个突变均导致蛋白质在比野生型蛋白质显著更低的盐酸胍浓度下展开，而基于其部分抑制上述两个突变的NUC-表型的能力分离出的三个抑制突变（sup）则导致在显著更高的盐酸胍浓度下展开。对具有两种不同氨基酸取代的核酸酶分子（nuc- + sup对或sup + sup对）的表征表明，通过ΔΔGD值测量，氨基酸取代对天然构象稳定性的影响可能不是恒定的，而是一个对同一分子中远处位点的其他取代的存在敏感的变量。令人惊讶的是，不同蛋白质之间log Kapp与盐酸胍浓度图的斜率变化高达35%。
