Nicholson H, Tronrud D E, Becktel W J, Matthews B W
Institute of Molecular Biology, Howard Hughes Medical Institute, Eugene, Oregon.
Biopolymers. 1992 Nov;32(11):1431-41. doi: 10.1002/bip.360321103.
It was previously shown that the two replacements Gly 77-->Ala (G77A) and Ala 82-->Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663-6667]. To further test this approach, three additional replacements--G113A, K60P and A93P--have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5-1.0 A in the short alpha-helix, 108-113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild-type lysozyme. The results suggest that the procedure used to predict sites for both Xaa-->Pro and Gly-->Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures.
先前的研究表明，两个替换位点Gly 77→Ala（G77A）和Ala 82→Pro（A82P）可提高噬菌体T4溶菌酶在pH 6.5时的热稳定性。据推测，此类替换会限制未折叠蛋白的自由度，从而降低去折叠熵[B. W. 马修斯、H. 尼科尔森和W. J. 贝克特尔（1987年）《美国国家科学院院刊》第84卷，第6663 - 6667页]。为进一步验证此方法，构建了另外三个替换位点——G113A、K60P和A93P。基于模型构建，判断这三个替换位点中的每一个都并非最优，因为它们往往会与蛋白质的相邻部分产生不利的范德华接触。通过这些突变蛋白晶体结构中观察到的构象调整，证实了G113A和K60P存在此类接触。就G113A而言，包含替换位点的短α - 螺旋（108 - 113）中主链构象变化为0.5 - 1.0 Å。就K60P而言，吡咯烷环显示出应变迹象。发现这三个变体在pH 2.0和pH 6.5时的热稳定性都与野生型溶菌酶非常接近。结果表明，用于预测Xaa→Pro和Gly→Ala位点的方法原则上是正确的。同时，这种类型替换预期的稳定性增加幅度较小，并且很容易被引入丙氨酸或脯氨酸所带来的应变抵消。这意味着用于选择能提高热稳定性的替换位点的标准至少必须严格。就T4溶菌酶而言，这严重限制了位点数量。分析揭示了脯氨酸前一个残基预测的构象能量表面与晶体结构中观察到的构象之间存在显著差异。
