Sengupta Kheya, Limozin Laurent, Tristl Matthias, Haase Ilka, Fischer Markus, Sackmann Erich
Lehrstuhl für Biophysik, E22, Technische Universität München, James-Franck-Strasse 1, D-85748 Garching, Germany.
Langmuir. 2006 Jun 20;22(13):5776-85. doi: 10.1021/la053310+.
We report the assembly of protein supramolecular structures at an air-water interface and coupling of artificial actin cortices to such structures. The coupling strategies adopted include electrostatic binding of actin to monolayers doped with lipids, exposing positively charged poly(ethylene glycol) headgroups; binding of biotinylated actin to lipids carrying biotin headgroups through avidin; binding of actin to membranes through biotinylated hisactophilin (a cellular actin-membrane coupler) using an avidin-biotin linkage; and coupling of actin to membranes carrying chelating lipids through a 15-nm-diameter protein capsid (bacterial lumazine synthase or LuSy) exhibiting histidine tags (which bind both to actin and to the chelating lipid). The distribution of the proteins in a direction normal to the interface was measured by neutron reflectivity under different conditions of pH and ionic strength. In the case of the first three binding methods, the thickness of the actin film was found to correspond to a single actin filament. Multilayers of actin could be formed only by using the multifunctional LuSy couplers that exhibit 60 hexahistidine tags and can thus act as actin cross-linkers. The LuSy-mediated binding can be reversibly switched by pH variations.
我们报道了在空气-水界面上蛋白质超分子结构的组装以及人工肌动蛋白皮层与此类结构的耦合。所采用的耦合策略包括：肌动蛋白与掺杂脂质的单层膜通过静电结合，暴露带正电荷的聚乙二醇头部基团；生物素化的肌动蛋白通过抗生物素蛋白与携带生物素头部基团的脂质结合；肌动蛋白通过抗生物素蛋白-生物素连接，经生物素化的组氨酸亲和蛋白（一种细胞肌动蛋白-膜耦合器）与膜结合；以及肌动蛋白通过展示组氨酸标签（可同时结合肌动蛋白和螯合脂质）的直径为15纳米的蛋白质衣壳（细菌核黄素合酶或LuSy）与携带螯合脂质的膜耦合。在不同的pH和离子强度条件下，通过中子反射率测量了蛋白质在垂直于界面方向上的分布。在前三种结合方法中，发现肌动蛋白膜的厚度对应于单根肌动蛋白丝。只有使用具有60个六组氨酸标签、因此可作为肌动蛋白交联剂的多功能LuSy耦合器，才能形成肌动蛋白多层膜。LuSy介导的结合可通过pH变化可逆地切换。
