Strategies for selection from protein libraries composed of de novo designed secondary structure modules
Strategies for selection from protein libraries composed of de novo designed secondary structure modules
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DOI：
10.1023/b:orig.0000009836.86519.eb
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发表时间：
2004-02-01
期刊：
影响因子：
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通讯作者：
Plückthun, A
中科院分区：
文献类型：
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作者：
Matsuura, T;Plückthun, A
随着越来越多的蛋白质结构被确定，很明显，自然界中只有有限数量的蛋白质折叠。为了探索自然界中发现的蛋白质折叠是否是蛋白质折叠问题的唯一解决方案，或者缺乏进化压力导致发现的不同蛋白质折叠的缺乏，我们开始构建蛋白质文库，而不受拓扑结构的任何限制。我们主要基于极性和非极性残基的模式化产生了平均长度为100个氨基酸残基的不同文库（所有α-螺旋、所有β-链和α-螺旋加β-链），其由不同比例的设计的二级结构模块（α-螺旋、β-链和β-转角）组成。通过对从文库中随机选择的蛋白质的分析，我们发现相当一部分纯α-螺旋蛋白质显示出与天然蛋白质相似的性质。使用这些库作为起点，我们的目标是建立一个选择系统，使我们能够从库中富集具有良好折叠特性（非聚集性，复合折叠）的蛋白质。我们已经开发了这样一种基于核糖体展示的方法。这种选择基于两个概念：（1）错误折叠的蛋白质对蛋白水解更敏感，（2）错误折叠和/或聚集的蛋白质更疏水。我们表明，通过应用这些选择标准中的每一个，可以富集在不溶性和无规卷曲蛋白质的蛋白质，是复杂折叠和可溶性的。
As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries ( all alpha-helix, all beta-strand and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand and beta-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure alpha-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, ( 2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.