Carra J H, Anderson E A, Privalov P L
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Biochemistry. 1994 Sep 6;33(35):10842-50. doi: 10.1021/bi00201a035.
Using microcalorimetry, we found an equilibrium intermediate state during the denaturation of the wild-type and five mutant staphylococcal nuclease proteins: V66L, V66W, G88V, D77A, and E75V. The presence of two distinct heat absorption peaks allowed direct measurement of the enthalpy differences between the native, intermediate, and denatured states. Conditions of low pH and high NaCl concentration facilitated observation of the intermediate, or I-state. We propose to consider the nuclease protein as composed of two subdomains, divided along the active-site cleft. The structure of the I-state apparently consists mainly of the folded beta-barrel subdomain, as does that of a nuclease fragment protein [Shortle, D., & Abeygunawardana, C. (1993) Structure 1, 121-134]. The cooperativity of folding of the subdomains is maintained by electrostatic bonds across the active-site cleft. Removal of these bonds by the mutation D77A or E75V results in decooperation of the protein's structure and a three-state mechanism of denaturation at pH 7.0. The origins of differences in the enthalpy change of denaturation and in the m value of guanidinium chloride-induced denaturation with mutant nucleases are discussed in terms of this three-state mechanism.
通过微量量热法，我们在野生型和五种突变型葡萄球菌核酸酶蛋白（V66L、V66W、G88V、D77A和E75V）变性过程中发现了一种平衡中间态。两个不同的吸热峰的存在使得能够直接测量天然态、中间态和变性态之间的焓差。低pH和高NaCl浓度的条件有助于观察中间态，即I态。我们建议将核酸酶蛋白视为由两个亚结构域组成，沿着活性位点裂隙划分。I态的结构显然主要由折叠的β桶亚结构域组成，核酸酶片段蛋白的结构也是如此[肖特尔，D.，& 阿贝古纳瓦德纳，C.（1993年）《结构》1，121 - 134]。亚结构域折叠的协同性通过活性位点裂隙上的静电键得以维持。通过D77A或E75V突变去除这些键会导致蛋白质结构的去协同作用以及在pH 7.0时的三态变性机制。根据这种三态机制讨论了突变型核酸酶变性焓变差异以及氯化胍诱导变性的m值差异的起源。
