ABSTRACT
From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC(14:0)), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC(12:0), diC(14:0) and diC(16:0)) and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidyl-glycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bhickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array. Secondly, we have previously deduced the entire conformation of retinal within the bacteriorhodopsin binding pocket in oriented purple membrane fragments. Using solid state deuterium NMR of the specifically deutero-methylated retinal labelled at each of the methyl positions in the molecule, the C-CD(3) bond vectors of the chromophore have been resolved to +/- 2 degrees . The ring conformation is 6-S-trans, but the polyene chain is slightly curved when in the protein binding site. Here, we describe studies on the protein in both the ground state and the trapped M(412)-state of the photocycle, to show that the orientation of the central methyl group (C(19)) on the polyene chain, which is at 40 degrees +/- 1 degrees with respect to the membrane normal, only changes its orientation by approximately 4 degrees upon 13-cis-isomerization. Thus, it is the Schiff base end of the chromophore which moves upon light incidence acting as a local switch on the protein in the photocycle, whilst the ring end of the chromophore moves rather less.
ABSTRACT
The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.
