Carra J H, Privalov P L
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Biochemistry. 1995 Feb 14;34(6):2034-41. doi: 10.1021/bi00006a025.
In a continuation of an earlier study [Carra, J., Anderson, E., & Privalov, P. (1994) Biochemistry 33, 10842-10850], we used differential scanning calorimetry to measure the enthalpy and heat capacity changes of denaturation for 11 mutant forms of staphylococcal nuclease, including the triple mutant [V66L+G88V+G79S]. Several mutant proteins with m- characteristics of guanidinium chloride denaturation were found to denature via a three-state mechanism with increasing temperature. Enthalpy changes for the transitions from the native to intermediate and from the intermediate to denatured states were determined. In the case of the triple mutant, the enthalpy of the second endothermic transition is greater than that of the first. Observation of this second transition provides an explanation for the previously reported large changes in the delta H denaturation for the triple mutant versus wild-type nuclease. The sequence specificity of structure in the intermediate state is discussed with relevance to m values of guanidinium chloride denaturation. The enthalpic level of the intermediate state depends upon the amino acid sequence, suggesting that stabilizing mutations can increase the extent or cohesion of structure present in the intermediate.
在早期一项研究[Carra, J., Anderson, E., & Privalov, P. (1994) Biochemistry 33, 10842 - 10850]的后续研究中，我们使用差示扫描量热法来测量11种葡萄球菌核酸酶突变体形式变性过程中的焓变和热容变化，其中包括三突变体[V66L + G88V + G79S]。发现几种具有氯化胍变性特征的突变蛋白随着温度升高通过三态机制变性。测定了从天然态到中间态以及从中间态到变性态转变的焓变。对于三突变体，第二个吸热转变的焓大于第一个。观察到的这第二个转变为先前报道的三突变体与野生型核酸酶相比在变性ΔH方面的巨大变化提供了解释。讨论了中间态结构的序列特异性与氯化胍变性的m值的相关性。中间态的焓水平取决于氨基酸序列，这表明稳定化突变可以增加中间态中存在的结构的程度或凝聚性。
