Slovic Avram M, Kono Hidetoshi, Lear James D, Saven Jeffery G, DeGrado William F
Department of Biochemistry and Molecular Biophysics, Johnson Foundation, School of Medicine, Makineni Theoretical Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1828-33. doi: 10.1073/pnas.0306417101. Epub 2004 Feb 6.
Although the interiors of membrane and water-soluble proteins are similar in their physicochemical properties, membrane proteins differ in having larger fractions of hydrophobic residues on their exteriors. Thus, it should be possible to water-solubilize membrane proteins by mutating their lipid-contacting side chains to more polar groups. Here, a computational approach was used to generate water-soluble variants of the potassium channel KcsA. As a probe of the correctness of the fold, the proteins contain an agitoxin2 binding site from a mammalian homologue of the channel. The resulting proteins express in high yield in Escherichia coli and share the intended functional and structural properties with KcsA, including secondary structure, tetrameric quaternary structure, and tight specific binding to both agitoxin2 and a small molecule channel blocker.
尽管膜蛋白和水溶性蛋白的内部在物理化学性质上相似，但膜蛋白的不同之处在于其外部有更大比例的疏水残基。因此，通过将与脂质接触的侧链突变为极性更强的基团，应该有可能使膜蛋白水溶性化。在此，采用了一种计算方法来生成钾通道KcsA的水溶性变体。作为折叠正确性的探针，这些蛋白质含有来自该通道哺乳动物同源物的阿吉毒素2结合位点。所得蛋白质在大肠杆菌中高产表达，并与KcsA具有预期的功能和结构特性，包括二级结构、四聚体四级结构以及与阿吉毒素2和小分子通道阻滞剂的紧密特异性结合。
