ENHANCED PROTEIN THERMOSTABILITY FROM SITE-DIRECTED MUTATIONS THAT DECREASE THE ENTROPY OF UNFOLDING
ENHANCED PROTEIN THERMOSTABILITY FROM SITE-DIRECTED MUTATIONS THAT DECREASE THE ENTROPY OF UNFOLDING
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DOI：
10.1073/pnas.84.19.6663
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发表时间：
1987-10-01
影响因子：
11.1
通讯作者：
BECKTEL, WJ
中科院分区：
文献类型：
--
作者：
MATTHEWS, BW;NICHOLSON, H;BECKTEL, WJ
有人提出，通过选定的氨基酸取代可以减少蛋白质的稳定性，从而减少展开的构型熵。两个这样的替换，其中一种是XAA .fwdarw。 pro和另一种形式的gly .fwdarw。 XAA在与已知的三维结构一致的位点的噬菌体T4溶菌酶中构建。两种取代都可以在生理pH值下稳定蛋白质的可逆和不可逆热变性。替代对酶活性没有影响。含脯氨酸的突变蛋白（ALA-82.fwdarw。Pro）的高分辨率晶体学分析表明，其三维结构与野生型酶基本相同。尽管在改变的氨基酸的附近存在局部构象调节，但其他突变酶（Gly-77.fwdarw。Ala）的总体结构也与野生型溶菌酶非常相似。许多此类氨基酸替代品的组合，每种氨基酸替代品都预计会贡献.apprxeq。 1 kcal/mol（1 cal = 4.184 J）到折叠的自由能，可以为蛋白质稳定性的实质性提高提供一般策略。
It is proposed that the stability of a protein can be incresed by selected amino acid substitutions that decrease the configurational entropy of unfolding. Two such substitutions, one of the form Xaa .fwdarw. Pro and the other of the form Gly .fwdarw. Xaa, were constructed in bacteriophage T4 lysozyme at sites consistent with the known three-dimensional structure. Both substitutions stabilize the protein toward reversible and irreversible thermal denaturation at physiological pH. The substitutions have no effect on enzymatic activity. High-resolution crystallographic analysis of the proline-containing mutant protein (Ala-82 .fwdarw. Pro) shows that its three-dimensional structure is essentially identical with the wild-type enzyme. The overall structure of the other mutant enzyme (Gly-77 .fwdarw. Ala) is also very similar to wild-type lysozyme, although there are localized conformational adjustments in the vicinity of the altered amino acid. The combination of a number of such amino acid replacements, each of which is expected to contribute .apprxeq. 1 kcal/mol (1 cal = 4.184 J) to the free energy of folding, may provide a general strategy for substantial improvement in the stability of a protein.