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首页> 外文期刊>Journal of Molecular Biology >Quantifying beta-sheet stability by phage display.
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Quantifying beta-sheet stability by phage display.

机译：通过噬菌体展示定量β-折叠稳定性。

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The small immunoglobulin G (IgG)-binding protein GB1 is a favored model system for the study of individual residue contributions to the stability of beta-sheets. Nevertheless, only a few of the many possible combinations of mutations have been characterized, leaving many questions unanswered. In order to allow the simultaneous evaluation of libraries of mutants, we have adapted a phage-display method, called shotgun scanning. This method combines a binding (i.e. stability) selection with high-throughput sequence analysis. Relative folding free energies determined from GB1-phage sequence data agree well with published GB1 thermal stability studies, validating the use of phage display to conduct quantitative stability studies on GB1, and further suggesting that this method is generally applicable to mutational analysis of protein stability. Examination of residue pairing in our large collection of GB1 mutants indicates that specific side-chain-side-chain interactions are much less important to beta-sheet stability than individual residue contributions. The discrepancy between this observation and published studies can be traced to anomalous stability of the alanine-substituted GB1 variants typically used as reference states in double mutant-cycle analyses. Finally, the combination of large library sizes and a quantitative stability selection should allow phage-based "computation" to be applied to protein design problems.
机译：小免疫球蛋白G（IgG）结合蛋白GB1是用于研究单个残基对β-折叠稳定性的贡献的模型系统。然而，仅对许多可能的突变组合中的少数进行了表征，从而使许多问题仍未得到解答。为了允许同时评估突变体文库，我们采用了一种噬菌体展示方法，称为shot弹枪扫描。该方法结合了结合（即稳定性）选择和高通量序列分析。由GB1噬菌体序列数据确定的相对折叠自由能与已发表的GB1热稳定性研究非常吻合，验证了使用噬菌体展示对GB1进行定量稳定性研究，进一步表明该方法通常适用于蛋白质稳定性的突变分析。在我们大量的GB1突变体中检查残基配对表明，特定的侧链-侧链相互作用对β-折叠稳定性的重要性远不如单个残基贡献重要。该观察结果与已发表的研究之间的差异可以追溯到丙氨酸取代的GB1变体的异常稳定性，该变体通常在双突变周期分析中用作参考状态。最后，大文库大小和定量稳定性选择的结合应允许将基于噬菌体的“计算”应用于蛋白质设计问题。

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