Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane
Mutational scanning reveals the determinants of protein insertion and association energetics in the plasma membrane
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DOI：
10.7554/elife.12125
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发表时间：
2016-01-29
期刊：
影响因子：
7.7
通讯作者：
Fleishman, Sarel Jacob
中科院分区：
文献类型：
--
作者：
Elazar, Assaf;Weinstein, Jonathan;Fleishman, Sarel Jacob
形成螺旋的片段插入细胞膜及其相互作用决定了所有质膜蛋白的结构、功能和表达水平。然而，膜蛋白能量学的系统和可靠的量化一直是具有挑战性的。我们开发了一种深度突变扫描方法来监测数百个点突变对细菌内膜内螺旋插入和自结合的影响。该分析定量了所有天然氨基酸在膜上27个位置的插入能，揭示了生物膜的疏水性明显高于人们所认识的。我们进一步定量了细胞质-膜界面上带正电荷的残基对膜-蛋白插入的贡献，并揭示了这些残基之间巨大的和意想不到的差异。最后，我们得到了糖蛋白A和ErbB2癌基因膜域的全面突变图景，发现插入和自结合在受体同源二聚体中强烈耦合。
Insertion of helix-forming segments into the membrane and their association determines the structure, function, and expression levels of all plasma membrane proteins. However, systematic and reliable quantification of membrane-protein energetics has been challenging. We developed a deep mutational scanning method to monitor the effects of hundreds of point mutations on helix insertion and self-association within the bacterial inner membrane. The assay quantifies insertion energetics for all natural amino acids at 27 positions across the membrane, revealing that the hydrophobicity of biological membranes is significantly higher than appreciated. We further quantitate the contributions to membrane-protein insertion from positively charged residues at the cytoplasm-membrane interface and reveal large and unanticipated differences among these residues. Finally, we derive comprehensive mutational landscapes in the membrane domains of Glycophorin A and the ErbB2 oncogene, and find that insertion and selfassociation are strongly coupled in receptor homodimers.