Fleming K G, Engelman D M
Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218, USA.
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14340-4. doi: 10.1073/pnas.251367498. Epub 2001 Nov 27.
The folding, stability, and oligomerization of helical membrane proteins depend in part on a precise set of packing interactions between transmembrane helices. To understand the energetic principles of these helix-helix interactions, we have used alanine-scanning mutagenesis and sedimentation equilibrium analytical ultracentrifugation to quantitatively examine the sequence dependence of the glycophorin A transmembrane helix dimerization. In all cases, we found that mutations to alanine at interface positions cost free energy of association. In contrast, mutations to alanine away from the dimer interface showed free energies of association that are insignificantly different from wild-type or are slightly stabilizing. Our study further revealed that the energy of association is not evenly distributed across the interface, but that there are several "hot spots" for interaction including both glycines participating in a GxxxG motif. Inspection of the NMR structure indicates that simple principles of protein-protein interactions can explain the changes in energy that are observed. A comparison of the dimer stability between different hydrophobic environments suggested that the hierarchy of stability for sequence variants is conserved. Together, these findings imply that the protein-protein interaction portion of the overall association energy may be separable from the contributions arising from protein-lipid and lipid-lipid energy terms. This idea is a conceptual simplification of the membrane protein folding problem and has implications for prediction and design.
螺旋膜蛋白的折叠、稳定性和寡聚化部分取决于跨膜螺旋之间精确的一组堆积相互作用。为了理解这些螺旋-螺旋相互作用的能量原理，我们使用丙氨酸扫描诱变和沉降平衡分析超速离心来定量研究血型糖蛋白A跨膜螺旋二聚化的序列依赖性。在所有情况下，我们发现界面位置突变为丙氨酸会消耗结合自由能。相比之下，远离二聚体界面的丙氨酸突变显示出与野生型无显著差异或略有稳定作用的结合自由能。我们的研究进一步表明，结合能并非均匀分布在整个界面上，而是存在几个相互作用的“热点”，包括参与GxxxG基序的甘氨酸。对核磁共振结构的检查表明，蛋白质-蛋白质相互作用的简单原理可以解释所观察到的能量变化。不同疏水环境中二聚体稳定性的比较表明，序列变体的稳定性层次是保守的。这些发现共同表明，总结合能中蛋白质-蛋白质相互作用部分可能与蛋白质-脂质和脂质-脂质能量项产生的贡献是可分离的。这个想法是膜蛋白折叠问题的概念简化，对预测和设计具有启示意义。
