SUBUNIT STOICHIOMETRY OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN IN CRYSTALS AND ON MEMBRANES - A HEPTAMERIC TRANSMEMBRANE PORE
SUBUNIT STOICHIOMETRY OF STAPHYLOCOCCAL ALPHA-HEMOLYSIN IN CRYSTALS AND ON MEMBRANES - A HEPTAMERIC TRANSMEMBRANE PORE
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DOI：
10.1073/pnas.91.26.12828
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发表时间：
1994-12-20
影响因子：
11.1
通讯作者：
BAYLEY, H
中科院分区：
文献类型：
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作者：
GOUAUX, JE;BRAHA, O;BAYLEY, H
阐明寡聚膜蛋白的准确亚基化学计量充满了复杂性。化学交联、分析超速离心、凝胶过滤和低分辨率电子显微镜研究的解释通常是不明确的。葡萄球菌 α-溶血素 (α HL) 是一种在细胞膜中形成通道的同源寡聚毒素，据信具有围绕六重对称轴排列的六个亚基。在这里，我们报告 X 射线衍射数据分析和化学修饰实验表明 α HL 寡聚物是七聚物。使用 α HL 低聚物单晶的 X 射线衍射数据计算的自旋转函数显示出七重旋转对称轴。通过电泳分离由带有野生型 α HL 电荷的亚基和通过单半胱氨酸突变体的靶向化学修饰产生的带有额外负电荷的亚基形成的 α HL 异聚体，确定兔红细胞膜上形成的 α HL 孔是七聚体。这些数据确定了三维晶体和生物膜上 α HL 跨膜孔的七聚寡聚状态。
Elucidation of the accurate subunit stoichiometry of oligomeric membrane proteins is fraught with complexities. The interpretations of chemical cross-linking, analytical ultracentrifugation, gel filtration, and low-resolution electron microscopy studies are often ambiguous. Staphylococcal alpha-hemolysin (alpha HL), a homooligomeric toxin that forms channels in cell membranes, was believed to possess six subunits arranged around a sixfold axis of symmetry. Here, we report that analysis of x-ray diffraction data and chemical modification experiments indicate that the alpha HL oligomer is a heptamer. Self-rotation functions calculated using x-ray diffraction data from single crystals of alpha HL oligomers show a sevenfold axis of rotational symmetry. The alpha HL pore formed on rabbit erythrocyte membranes was determined to be a heptamer by electrophoretic separation of alpha HL heteromers formed from subunits with the charge of wild-type alpha HL and subunits with additional negative charge generated by targeted chemical modification of a single-cysteine mutant. These data establish the heptameric oligomerization state of the alpha HL transmembrane pore both in three-dimensional crystals and on a biological membrane.