THE EFFECT OF TEMPERATURE AND PROTEIN-CONTENT ON THE DISPERSIVE PROPERTIES OF BACTERIORHODOPSIN FROM H-HALOBIUM IN RECONSTITUTED DMPC COMPLEXES FREE OF ENDOGENOUS PURPLE MEMBRANE-LIPIDS - A FREEZE-FRACTURE ELECTRON-MICROSCOPY STUDY

Ten reconstituted bilayer complexes of bacteriorhodopsin from Halobacterium halobium and 1,2-dimyristoyl-sn-glycerol-3- phosphocholine with protein/lipid mole ratios of between 1:1440 and 1:67, have been entirely free (less than 0.02%) of endogenous purple membrane phospholipid as judged by 31P-NMR methods using Triton X-100 and cholate as detergents for solubilization of the protein and reconstitution by detergent dialysis. The nitroxide spin-label Tempo has been employed to determine the bilayer gel to liquid-crystalline phase transition temperature of the reconstituted complexes which was shown to broaden with increasing protein content, but remain centered at 23-24°C. Freeze-fracture electron micrographs of the recombinants showed that the arrangement of the protein particles depended upon the temperature from which the complexes were quenched for study, the protein content of the complexes as well as the rate of freezing. In recombinants quenched from below the bilayer phase transition temperature, protein particles were restricted to areas in the bilayers of low structural order at dilute protein content but randomly dispersed at high protein content, regardless of the rate of sample freezing. When quenched from above the bilayer phase transition temperature, complexes quenched at a faster rate of freezing produced randomly dispersed particles whilst a slower rate of freezing produced areas devoid of protein particles. No indication of the characteristic hexagonal packing of bacteriorhodopsin molecules, as reported for the purple membrane (Blaurock, A.E. and Stoeckenius, W. (1971) Nature, 233, 152-155) or reconstituted complexes still containing endogenous purple membrane phospholipids (Cherry, R.J. et al. (1978) J. Mol. Biol. 121, 282-298), were observed. The results are interpreted in terms of a lipid-mediated promotion for bacteriorhodopsin association into the hexagonal lattice, possibly through association with the negatively charged lipids of the purple membrane at the bilayer surface. © 1989