Single-molecule imaging of cooperative assembly of γ-hemolysin on erythrocyte membranes
Single-molecule imaging of cooperative assembly of γ-hemolysin on erythrocyte membranes
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DOI：
10.1093/emboj/cdg498
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发表时间：
2003-10-01
期刊：
影响因子：
11.4
通讯作者：
Higuchi, H
中科院分区：
文献类型：
--
作者：
Nguyen, VT;Kamio, Y;Higuchi, H
使用单分子荧光成像研究金黄色葡萄球菌 LukF 和 HS 单体在红细胞膜上组装成成孔低聚物（γ-溶血素）的情况。我们将杂低聚物与单体区分开来，如附着在单体亚基上的不同染料之间的荧光共振能量转移所示。低聚物中 LukF（供体）和 HS（受体）亚基的化学计量分别是根据能量转移过程中的受体发射强度和直接受体激发推导出来的。基于单体和寡聚中间体的群体，我们估计了组装途径的 11 个连续平衡常数，从单体的膜结合开始，通过单孔寡聚化，最终形成孔簇。多个阶段高度协作，极大地提高了装配效率。
Single-molecule fluorescence imaging was used to investigate assembly of Staphylococcus aureus LukF and HS monomers into pore-forming oligomers (gamma-hemolysin) on erythrocyte membranes. We distinguished the hetero-oligomers from the monomers, as indicated by fluorescence resonance energy transfer between different dyes attached to monomeric subunits. The stoichiometry of LukF (donor) and HS (acceptor) subunits in oligomers was deduced from the acceptor emission intensities during energy transfer and by direct acceptor excitation, respectively. Based on populations of monomeric and oligomeric intermediates, we estimated 11 sequential equilibrium constants for the assembly pathway, beginning with membrane binding of monomers, proceeding through single pore oligomerization, and culminating in the formation of clusters of pores. Several stages are highly cooperative, critically enhancing the efficiency of assembly.