Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.
Glycophorin A dimerization is driven by specific interactions between transmembrane alpha-helices.
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DOI：
10.1016/s0021-9258(18)42569-0
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发表时间：
1992-04
期刊：
影响因子：
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通讯作者：
Mark A. LemmonS;J. Flanagan;J. Hunt;B. Adair;Barbara-Jean Bormanng;Christopher E. Dempseyll;D. Engelman
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文献类型：
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作者：
Mark A. LemmonS;J. Flanagan;J. Hunt;B. Adair;Barbara-Jean Bormanng;Christopher E. Dempseyll;D. Engelman
跨膜α -螺旋之间的特异性并排相互作用可能在整体膜蛋白的组装和功能中起重要作用。我们描述了一个系统的遗传和生物物理分析这些相互作用。感兴趣的跨膜α -螺旋结构域融合到葡萄球菌核酸酶的c端。由此产生的嵌合体可以在大肠杆菌中高水平表达，并且易于纯化。在我们最初的应用中，我们研究了人糖蛋白A （GpA）的单跨膜α -螺旋，被认为介导了该蛋白的sds稳定二聚化。由此产生的嵌合体在SDS中形成二聚体，当加入与GpA跨膜结构域相对应的肽时，该二聚体被破坏。缺失诱变已被用来描述这种行为所需的最小跨膜结构域。位点特异性诱变表明，以前作为潜在界面残基的蛋氨酸残基可以被其他疏水残基取代，而不会破坏二聚体。相比之下，在α -螺旋的不同面上的缬氨酸的相当保守的取代破坏了二聚化，这表明在螺旋-螺旋相互作用中具有高度的特异性。这种方法允许定义相互作用的螺旋之间的接口。
Specific side-by-side interactions between transmembrane alpha-helices may be important in the assembly and function of integral membrane proteins. We describe a system for the genetic and biophysical analysis of these interactions. The transmembrane alpha-helical domain of interest is fused to the C-terminus of staphylococcal nuclease. The resulting chimera can be expressed at high levels in Escherichia coli and is readily purified. In our initial application we study the single transmembrane alpha-helix of human glycophorin A (GpA), thought to mediate the SDS-stable dimerization of this protein. The resulting chimera forms a dimer in SDS, which is disrupted upon addition of a peptide corresponding to the transmembrane domain of GpA. Deletion mutagenesis has been used to delineate the minimum transmembrane domain sufficient for this behavior. Site-specific mutagenesis shows that a methionine residue, previously implicated as a potential interfacial residue, can be replaced with other hydrophobic residues without disrupting dimerization. By contrast, rather conservative substitutions at a valine on a different face of the alpha-helix disrupt dimerization, suggesting a high degree of specificity in the helix-helix interactions. This approach allows the interface between interacting helices to be defined.