Solubilization of a membrane protein by combinatorial supercharging.
Solubilization of a membrane protein by combinatorial supercharging.
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通过组合增压溶解膜蛋白。
DOI：
10.1021/cb1001729
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发表时间：
2011
影响因子：
4
通讯作者：
Weiss,GregoryA
中科院分区：
文献类型：
--
作者：
Hajduczki,Agnes;Majumdar,Sudipta;Fricke,Marie;Brown,IsolaAM;Weiss,GregoryA
疏水和聚集倾向，膜蛋白往往被证明太不溶常规的体外生化研究。为了设计人小洞蛋白-1的可溶变体，噬菌体展示的小洞蛋白变体库通过替换带电残基靶向疏水膜内结构域。不溶性的抗选择去除疏水变异，而与已知的小窝蛋白配体HIV gp41结合的阳性选择分离出功能性折叠变异。与几个小洞蛋白结合伙伴的实验表明，从文库中选择的可溶解的全长小洞蛋白变体成功折叠和功能。这种小洞蛋白变体可以检测小洞蛋白和小洞蛋白之间的直接相互作用。聚集在一个假定的螺旋的一个面，溶解突变提示一个结构模型的膜内结构域的小窝蛋白。该方法为通过噬菌体展示实现膜相关蛋白的溶解和工程提供了一种潜在的通用方法。
Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventionalin vitrobiochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.