Nishimura C, Uversky V N, Fink A L
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Biochemistry. 2001 Feb 20;40(7):2113-28. doi: 10.1021/bi000861k.
The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.
在中性pH条件下，野生型和突变型葡萄球菌核酸酶（SNase）的稳定性和折叠动力学受到中等至高浓度盐的显著影响。观察到其对热变性和尿素变性的稳定性有非常显著的提高；例如，0.4M硫酸钠使野生型SNase的自由能增加了超过2千卡/摩尔。对于NCA SNase突变体，盐的存在消除了该变体在中性pH下观察到的冷变性，并显著提高了其稳定性。还观察到盐对重折叠动力学有显著影响。对于NCA SNase，盐的存在显著提高了折叠速率（高达5倍）。另一方面，特别是氯离子，大大降低了野生型蛋白质的折叠速率。由于脯氨酸异构化导致的慢相速率因盐而增加，这些步骤一定与构象过程相关。唯一的色氨酸（Trp140）与64位残基处的工程荧光受体之间的荧光能量转移表明，添加高浓度的KCl会导致形成一种在较低盐浓度下未观察到的瞬时折叠中间体，其中140和64位残基比天然状态下更接近。盐对折叠动力学的影响归因于瞬时折叠中间体稳定性的增强。由于高的等电点导致的高净电荷和相对较低的固有疏水性的结合，可能导致葡萄球菌核酸酶在中性pH下仅具有边缘稳定性。盐对葡萄球菌核酸酶的结构、稳定性和动力学的影响归因于抗衡离子即阴离子的结合，从而使分子内静电排斥最小化。如观察到的那样，这导致稳定性增加、结构更多和紧凑性更高。因此，SNase在中性pH下存在局部静电排斥，这可能是其边缘稳定性的原因。结果表明，一般来说，在具有大量净电荷的条件下，边缘稳定的球状蛋白质会被盐显著稳定。
