Young H S, Rigaud J L, Lacapère J J, Reddy L G, Stokes D L
Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York 10016, USA.
Biophys J. 1997 Jun;72(6):2545-58. doi: 10.1016/S0006-3495(97)78898-2.
In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation.
为了更好地确定控制膜蛋白重构和二维结晶的参数，我们研究了兔肌浆网中的Ca2(+)-ATP酶。这种离子泵在其天然膜中形成钒酸盐诱导的晶体，并且此前已在高脂质与蛋白质比例下进行重构以用于功能研究。我们已经对低脂质与蛋白质比例下纯化的Ca2(+)-ATP酶的重构进行了表征，并发现了产生适合螺旋重构的长管状晶体的方法。使用C12E8（正十二烷基-八乙二醇单醚）完全溶解脂质和纯化的Ca2(+)-ATP酶的各种混合物，然后使用BioBeads去除C12E8。缓慢去除导致形成两类囊泡，通过蔗糖密度梯度将蛋白脂质体群体与脂质体群体分离。这些蛋白脂质体的脂质与蛋白质比例为1:2，通过异硫氰酸荧光素标记确定，实际上100%的分子面向囊泡外部。冻融循环导致这些蛋白脂质体大量聚集，如果包含磷脂酰乙醇胺和磷脂酸，或者如果双层中掺杂少量C12E8，钒酸盐诱导的管状晶体就会从聚集体中生长出来。因此，我们的方法包括两个步骤——重构后结晶——使我们能够分别考虑双层形成机制与结晶和管形成机制。
