Quantifying β-sheet stability by phage display
Quantifying β-sheet stability by phage display
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DOI：
10.1016/s0022-2836(02)00738-6
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发表时间：
2002-09-06
影响因子：
5.6
通讯作者：
Cochran, AG
中科院分区：
文献类型：
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作者：
Distefano, MD;Zhong, A;Cochran, AG
小免疫球蛋白G （IgG）结合蛋白GB1是研究个体残基对β -片稳定性贡献的理想模型系统。然而，在许多可能的突变组合中，只有少数被确定了特征，留下了许多未解之谜。为了能够同时评估突变体文库，我们采用了一种噬菌体展示方法，称为散弹枪扫描。该方法结合了结合（即稳定性）选择和高通量序列分析。由GB1噬菌体序列数据测定的相对折叠自由能与已发表的GB1热数据吻合较好。验证了利用噬菌体展示法对GB1进行定量稳定性研究，进一步表明该方法普遍适用于蛋白质稳定性的突变分析。在我们大量收集的GB1突变体中对残基配对的研究表明，特定的侧链-侧链相互作用对β -片稳定性的影响远不如单个残基的贡献重要。这一观察结果与已发表的研究之间的差异可以追溯到双突变周期分析中通常用作参考状态的丙氨酸取代的GB1变异的异常稳定性。最后，大型文库和定量稳定性选择的结合应该允许基于噬菌体的“计算”应用于蛋白质设计问题。(C) 2002 Elsevier Science Ltd.版权所有。
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. (C) 2002 Elsevier Science Ltd. All rights reserved.