The long-term objective of this research is to understand how the structure of a supramolecular membrane complex enables its function. The goal of this project is to reconstitute the photoreceptor complex (PRC) of photosynthetic bacteria from its fundamental compenents and to use our expertise in membrane biochemistry to systematically evaluate mechanistic relationships. This laboratory has been successful in reversibly dissociating the core light-harvesting complex (LH1) of four different photosynthetic bacteria and has characterized a subunit complex from each. Moreover, the subunit complex and lH1 of three of these bacteria have been reconstituted from their fundamental components. The methodology of reconstitution is especially powerful for obtaining detailed information since subtle changes can be made in the cofactors or protein and their effect on function measured. The role of specific amino acids in stabilizing the subunit and LH1 complexes in being explored by reconstitution experiments using aalpha- and/or beta-polypeptides polypeptides isolated from specifically prepared mutant bacteria. Highly conserved residues thought to be involved in hydrogen bonding interactions with BChl are among the first amino acids being targeted by site-directed mutagenesis. Chemical synthesis of a series of polypeptides related to the last 31 amino acids of the beta-polypeptide of Rb. sphaeroides will be pursued. This amino acid sequence was selected because our work has demonstrated that this truncated beta-polypeptide can be reconstituted to form a subunit complex as effectively as the native beta-polypeptide. The immediate goal of this approach is to evaluate the minimal length of the polypeptide required to form the subunit complex and the role of highly conserved amino acids starting from the C-terminal end of the beta- polypeptide and progressing towards the n-terminal end. Structure determination of the subunit complex by high resolution NMR will be attempted. Especially useful will be the application of the powerful methods of random deuteration, alternate C13-C12 labelling and chiral labelling that have recently been developing by one of our collaborators. The ability to reconstitute unique LH1 complexes and reassociate these with the reaction center will allow us to probe sites of association between these macromolecular membrane complexes which compose the PRC.
