pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state.
pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state.
复制标题
葡萄球菌核酸酶稳定性的 pH 依赖性：变性状态下大量静电相互作用的证据。
作者：
S. Whitten;B. García‐Moreno E.
用两种独立的平衡热力学方法研究了葡萄球菌核酸酶稳定性的pH依赖性。首先，通过用尿素（Delta）、GdnHCl（Delta）和热（Delta）进行荧光监测变性来测量在pH 9至3.5范围内的稳定性。其次，通过在天然（100 mM KCl）和解折叠（6.0 M GdnHCl）条件下电位测定的H（+）滴定曲线（Delta）的数值积分。Delta、Delta和Delta描述的稳定性的pH依赖性相当，但与Delta描述的pH依赖性显著不同。在pH 9和pH 4之间的Delta的降低比在相同pH范围内的Delta、Delta和Delta的降低大4 kcal/mol。在6 M GdnHCl中，所有可电离基团均用模型化合物的pK（a）值滴定。因此，Delta表示天然状态（N）与非结构化或扩展和高度屏蔽构象的集合之间的自由能差。相比之下，Delta和Delta在pH 9 - 5之间描述的稳定性的较浅pH依赖性与组氨酸在变性状态（D）下具有降低的天然样pK（a）值的滴定一致。这些降低的pK（a）值可能反映了与其他29个基本基团的长程静电相互作用，并且是D态的紧凑特性的结果。Δ和Δ在pH < 5时的急剧变化表明，接近pH 5时，D态的结构和热力学特征向酸性残基以正常pK（a）值滴定的状态转变，可能是因为与碱性残基的静电相互作用丢失，可能是膨胀的结果。
The pH dependence of stability of staphylococcal nuclease was studied with two independent equilibrium thermodynamic approaches. First, by measurement of stability in the pH range 9 to 3.5 by fluorescence-monitored denaturation with urea (Delta), GdnHCl (Delta), and heat (Delta). Second, by numerical integration of H(+) titration curves (Delta) measured potentiometrically under native (100 mM KCl) and unfolding (6.0 M GdnHCl) conditions. The pH dependence of stability described by Delta, Delta, and Delta was comparable but significantly different from the one described by Delta. The decrease in Delta between pH 9 and pH 4 was 4 kcal/mol greater than the decrease in Delta, Delta, and Delta in the same pH range. In 6 M GdnHCl, all the ionizable groups titrated with the pK(a) values of model compounds. Therefore, Delta represents the free energy difference between the native state (N) and an ensemble of unstructured, or expanded, and highly screened conformations. In contrast, the shallower pH dependence of stability described by Delta and by Delta between pH 9 and 5 was consistent with the titration of histidines with depressed, nativelike pK(a) values in the denatured state (D). These depressed pK(a) values likely reflect long-range electrostatic interactions with the other 29 basic groups and are a consequence of the compact character of the D state. The steep change in Delta and Delta at pH < 5 suggests that near pH 5 the structural and thermodynamic character of the D state shifts toward a state in which acidic residues titrate with normal pK(a) values, presumably because the electrostatic interactions with basic residues are lost, maybe as a consequence of an expansion.