Varadarajan R, Nagarajaram H A, Ramakrishnan C
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13908-13. doi: 10.1073/pnas.93.24.13908.
Temperature-sensitive (Ts) mutants of a protein are an extremely powerful tool for studying protein function in vivo and in cell culture. We have devised a method to predict those residues in a protein sequence that, when appropriately mutated, are most likely to give rise to a Ts phenotype. Since substitutions of buried hydrophobic residues often result in significant destabilization of the protein, our method predicts those residues in the sequence that are likely to be buried in the protein structure. We also indicate a set of amino acid substitutions, which should be made to generate a Ts mutant of the protein. This method requires only the protein sequence. No structural information or homologous sequence information is required. This method was applied to a test data set of 30 nonhomologous protein structures from the Protein Data Bank. All of the residues predicted by the method to be > or = 95% buried were, in fact, buried in the protein crystal structure. In contrast, only 50% of all hydrophobic residues in this data set were > or = 95% buried. This method successfully predicts several known Ts and partially active mutants of T4 lysozyme, lambda repressor, gene V protein, and staphylococcal nuclease. This method also correctly predicts residues that form part of the hydrophobic cores of lambda repressor, myoglobin, and cytochrome b562.
蛋白质的温度敏感（Ts）突变体是研究体内和细胞培养中蛋白质功能的极其强大的工具。我们设计了一种方法来预测蛋白质序列中的那些残基，当这些残基经过适当突变后，最有可能产生Ts表型。由于埋藏的疏水残基的替换常常导致蛋白质的显著不稳定，我们的方法预测序列中那些可能埋藏在蛋白质结构中的残基。我们还指出了一组氨基酸替换，应该进行这些替换以产生该蛋白质的Ts突变体。这种方法只需要蛋白质序列。不需要结构信息或同源序列信息。该方法应用于来自蛋白质数据库的30个非同源蛋白质结构的测试数据集。该方法预测为≥95%埋藏的所有残基实际上都埋藏在蛋白质晶体结构中。相比之下，该数据集中所有疏水残基中只有50%是≥95%埋藏的。该方法成功地预测了T4溶菌酶、λ阻遏物、基因V蛋白和葡萄球菌核酸酶的几个已知的Ts和部分活性突变体。该方法还正确地预测了构成λ阻遏物、肌红蛋白和细胞色素b562疏水核心一部分的残基。
