Hong Heedeok, Park Sangho, Jiménez Ricardo H Flores, Rinehart Dennis, Tamm Lukas K
Department of Molecular Physiology and Biological Physics, University of Virginia Health System, Charlottesville, VA 22908-0736, USA.
J Am Chem Soc. 2007 Jul 4;129(26):8320-7. doi: 10.1021/ja068849o. Epub 2007 Jun 12.
Aromatic residues are frequently found in helical and beta-barrel integral membrane proteins enriched at the membrane-water interface. Although the importance of these residues in membrane protein folding has been rationalized by thermodynamic partition measurements using peptide model systems, their contribution to the stability of bona fide membrane proteins has never been demonstrated. Here, we have investigated the contribution of interfacial aromatic residues to the thermodynamic stability of the beta-barrel outer membrane protein OmpA from Escherichia coli in lipid bilayers by performing extensive mutagenesis and equilibrium folding experiments. Isolated interfacial tryptophanes contribute -2.0 kcal/mol, isolated interfacial tyrosines contribute -2.6 kcal/mol, and isolated interfacial phenylalanines contribute -1.0 kcal/mol to the stability of this protein. These values agree well with the prediction from the Wimley-White interfacial hydrophobicity scale, except for tyrosine residues, which contribute more than has been expected from the peptide models. Double mutant cycle analysis reveals that interactions between aromatic side chains become significant when their centroids are separated by less than 6 A but are nearly insignificant above 7 A. Aromatic-aromatic side chain interactions are on the order of -1.0 to -1.4 kcal/mol and do not appear to depend on the type of aromatic residue. These results suggest that the clustering of aromatic side chains at membrane interfaces provides an additional heretofore not yet recognized driving force for the folding and stability of integral membrane proteins.
芳香族残基常见于富集于膜 - 水界面的螺旋和β - 桶状整合膜蛋白中。尽管通过使用肽模型系统的热力学分配测量已阐明了这些残基在膜蛋白折叠中的重要性，但它们对真正膜蛋白稳定性的贡献尚未得到证实。在此，我们通过进行广泛的诱变和平衡折叠实验，研究了界面芳香族残基对大肠杆菌β - 桶状外膜蛋白OmpA在脂质双层中热力学稳定性的贡献。孤立的界面色氨酸对该蛋白稳定性的贡献为 -2.0千卡/摩尔，孤立的界面酪氨酸为 -2.6千卡/摩尔，孤立的界面苯丙氨酸为 -1.0千卡/摩尔。除了酪氨酸残基外，这些值与Wimley - White界面疏水性标度的预测结果吻合良好，酪氨酸残基的贡献比肽模型预期的要大。双突变循环分析表明，当芳香族侧链质心间距小于6埃时，它们之间的相互作用变得显著，但在7埃以上时几乎不显著。芳香族 - 芳香族侧链相互作用的量级为 -1.0至 -1.4千卡/摩尔，且似乎不依赖于芳香族残基的类型。这些结果表明，膜界面处芳香族侧链的聚集为整合膜蛋白的折叠和稳定性提供了一种迄今尚未被认识到的额外驱动力。
