High-precision mapping of protein protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform.
High-precision mapping of protein protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform.
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DOI：
10.1093/nar/gkm563
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发表时间：
2007
影响因子：
14.9
通讯作者：
Savilahti, Harri
中科院分区：
文献类型：
--
作者：
Pajunen, Maria;Turakainen, Hilkka;Poussu, Eini;Peranen, Johan;Vihinen, Mauno;Savilahti, Harri
了解蛋白质-蛋白质相互作用的网络是全面描述细胞功能的重要组成部分。虽然有效的技术很容易用于相互作用的蛋白质伴侣的初始识别，但缺乏用于随后的蛋白质-蛋白质界面区域高分辨率映射的实用策略。我们在这里提出了一种遗传策略来精确地绘制氨基酸相互作用的蛋白质区域。该系统基于一个综合插入突变体库的并行构建、采样和分析。该方法整合了基于Mu的体外转位随机五肽突变蛋白，酵母双杂交筛选和高分辨率遗传足迹。该策略是通用的，适用于任何相互作用的蛋白质对。我们通过绘制人类JFC1中与Rab8A相互作用的区域来证明该方法的可行性，并表明这种关联是由Slp同源结构域1介导的。
Understanding networks of protein–protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein–protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1.