Insertion of bacteriorhodopsin into polymerized diacetylenic phosphatidylcholine bilayers

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.