Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
Biophys Chem. 2013 Oct-Nov;180-181:86-94. doi: 10.1016/j.bpc.2013.06.018. Epub 2013 Jul 1.
Six single substitution mutations, V66F, V66G, V66N, V66Q, V66S, V66T, and V66Y, were made in the background of a highly stable triple mutant (P117G, H124L, and S128A) of staphylococcal nuclease. The thermodynamic stabilities of wild type staphylococcal nuclease, of the stable triple mutant and of its six variants were determined by guanidine hydrochloride denaturation in thirteen different buffers spanning the pH range 4.5 to 10.2. Within experimental error the values of [Formula: see text] and mGuHCl for the various proteins measured over this wide range of pH maintain a constant offset from one another, tracing a series of approximately parallel curves. This data offers an independent means of determining the error of stabilities and slopes determined by guanidine hydrochloride denaturations and shows that previous error estimates are accurate. More importantly, this behavior cannot be reconciled with a three-state denaturation model for staphylococcal nuclease. The large variations in mGuHCl observed in these mutants must therefore arise from other causes.
在金黄色葡萄球菌核酸酶高度稳定的三重突变体（P117G、H124L 和 S128A）背景下，共产生了六个单一取代突变（V66F、V66G、V66N、V66Q、V66S、V66T 和 V66Y）。通过在 13 种不同的缓冲液中进行盐酸胍变性，测定了野生型金黄色葡萄球菌核酸酶、稳定的三重突变体及其六种变体的热力学稳定性，该 pH 范围为 4.5 至 10.2。在实验误差范围内，在如此宽的 pH 范围内测量的各种蛋白质的[Formula: see text]和 mGuHCl 值彼此保持恒定的偏移，描绘了一系列大致平行的曲线。这些数据为通过盐酸胍变性确定稳定性和斜率的误差提供了独立的方法，并表明先前的误差估计是准确的。更重要的是，这种行为不能与金黄色葡萄球菌核酸酶的三态变性模型相协调。因此，在这些突变体中观察到的 mGuHCl 的巨大变化必然是由其他原因引起的。
