ABSTRACT
The orientation of the light-driven chloride pump, halorhodopsin, in the membrane was determined using antibodies directed against a synthetic peptide which represents the C-terminal segment of the protein. Antibodies against this decapeptide did not bind to right-side-out cell vesicles. Partial inversion by sonication or lysis under low salt conditions exposed this COOH-terminal antigenic site. Antibody binding was removed by preincubation with the decapeptide. The COOH terminus of the molecule is therefore located on the cytoplasmic surface of the membrane.
Subject(s)
Bacteriorhodopsins/analysis , Cell Membrane/analysis , Halobacterium/analysis , Amino Acid Sequence , Cytoplasm/analysis , Enzyme-Linked Immunosorbent Assay , Halobacterium/ultrastructure , Halorhodopsins , Molecular Sequence Data , Protein ConformationABSTRACT
The topography of the light-harvesting polypeptides of Rhodopseudomonas viridis was investigated using cleavable chemical cross-linkers. To this end a set of succinimidyl esters and surface-specific sulfosuccinimidyl esters of different span widths were synthesized. The cross-linking reagents have been characterized using NMR and infrared spectroscopy and thin-layer chromatography. The cross-linking reaction was carried out under physiological conditions and the aggregates were analyzed by the methods of one- and two-dimensional polyacrylamide gel electrophoresis and by immunoblot analysis. We found cross-linkage between B1015-alpha and B1015-alpha, between B1015-alpha and B1015-beta and B1015-beta and B1015-beta. Aggregates of higher molecular mass were hetero-oligomers of B1015-alpha and B1015-beta containing three and four polypeptides, respectively. The results obtained in this work indicate a very tight contact among the light-harvesting polypeptides. We assume that the light-harvesting polypeptides are localized alternately as dimers of B1015-alpha and B1015-beta around the reaction centre core.
