COLD DENATURATION AND (H2O)-H-2 STABILIZATION OF A STAPHYLOCOCCAL NUCLEASE MUTANT
COLD DENATURATION AND (H2O)-H-2 STABILIZATION OF A STAPHYLOCOCCAL NUCLEASE MUTANT
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DOI：
10.1073/pnas.88.17.7715
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发表时间：
1991-09-01
影响因子：
11.1
通讯作者：
FINK, AL
中科院分区：
文献类型：
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作者：
ANTONINO, LC;KAUTZ, RA;FINK, AL
冷变性现在被认为是蛋白质的一般特性，但只在不稳定条件下观察到，例如中等变性剂浓度或低pH。通过使用定点诱变来破坏蛋白质的稳定，我们在没有变性剂的情况下观察到葡萄球菌核酸酶突变体在pH 7.0下的冷变性。我们将其称为NCA S28G，这是葡萄球菌核酸酶和豆豆蛋白a之间的一种杂交蛋白，其中存在点突变Ser-28 -> Gly。圆二色性（CD）测定的最大稳定温度（t(max)）为18.1℃，热展开转变中点（t(m)）为0.6℃和30.0℃。这些值可以与野生型葡萄球菌核酸酶在52.5℃的t(m)进行比较，在这些条件下没有观察到冷变性。当在(H2O)-H-2中通过核磁共振、CD或荧光检测突变体的稳定性时，可以注意到t（max）处折叠蛋白的数量大幅增加以及t（max）的减少，反映了稳定性的增加。
Cold denaturation is now recognized as a general property of proteins but has been observed only under destabilizing conditions, such as moderate denaturant concentration or low pH. By destabilizing the protein using site-directed mutagenesis, we have observed cold denaturation at pH 7.0 in the absence of denaturants in a mutant of staphylococcal nuclease, which we call NCA S28G for a hybrid protein between staphylococcal nuclease and concanavalin A in which there is the point mutation Ser-28 --> Gly. The temperature of maximum stability (t(max)) as determined by circular dichroism (CD) was 18.1-degrees-C, and the midpoints of the thermal unfolding transitions (t(m)) were 0.6-degrees-C and 30.0-degrees-C. These values may be compared with the t(m) of 52.5-degrees-C for wild-type staphylococcal nuclease, for which no cold denaturation was observed under these conditions. When the stability of the mutant was examined in (H2O)-H-2 by NMR, CD, or fluorescence, a substantial increase in the amount or folded protein at the t(max) was noted as well as a decrease in t(max), reflecting increased stability.