Ahl P L, Price R, Smuda J, Gaber B P, Singh A
Center for Bio/molecular Science and Engineering, Naval Research Laboratory, Washington, DC.
Biochim Biophys Acta. 1990 Oct 5;1028(2):141-53. doi: 10.1016/0005-2736(90)90148-h.
We have developed a method to incorporate the membrane protein bacteriorhodopsin into polymerized bilayers composed of a diacetylenic phosphatidylcholine, 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and a non-polymerizable phospholipid, dinonanoylphosphatidylcholine (DNPC). The extent of DC8,9PC polymerization in the bilayer was significantly improved when 2:1 mole ratio DNPC-DC8,9PC was used. Octyl glucopyranoside-solubilized bacteriorhodopsin was inserted into the polymerized DNPC-DC8,9PC bilayers by overnight incubation at 4 degrees C followed by dialysis to remove the detergent. The protein was inserted into the membranes after photo-polymerization to avoid inactivation of the protein due to the UV irradiation. The insertion of bacteriorhodopsin into the polymerized DNPC-DC8,9PC membranes was confirmed by density gradient centrifugation, UV/visible spectroscopy, and freeze fracture electron microscopy. The polymerized DNPC-DC8,9PC membranes containing bacteriorhodopsin were about 10% protein by weight. These results suggest that mixed lipid systems such as the DNPC-DC8,9PC can be used to improve both the extent of polymerization and the efficiency of membrane protein incorporation in the polymerized bilayer.
我们已经开发出一种方法，可将膜蛋白细菌视紫红质整合到由二乙炔基磷脂酰胆碱、1,2-双（二十三碳-10,12-二炔酰基）-sn-甘油-3-磷酸胆碱（DC8,9PC）和一种不可聚合的磷脂二壬酰磷脂酰胆碱（DNPC）组成的聚合双层膜中。当使用DNPC与DC8,9PC摩尔比为2:1时，双层膜中DC8,9PC的聚合程度显著提高。将辛基吡喃葡萄糖苷增溶的细菌视紫红质在4℃下孵育过夜，然后通过透析去除去污剂，从而插入到聚合的DNPC-DC8,9PC双层膜中。为避免蛋白质因紫外线照射而失活，在光聚合后将蛋白质插入膜中。通过密度梯度离心、紫外/可见光谱和冷冻断裂电子显微镜证实了细菌视紫红质插入到聚合的DNPC-DC8,9PC膜中。含有细菌视紫红质的聚合DNPC-DC8,9PC膜按重量计约含10%的蛋白质。这些结果表明，诸如DNPC-DC8,9PC之类的混合脂质体系可用于提高聚合双层膜中的聚合程度和膜蛋白整合效率。
