The long term objective of this work is to learn how integral membrane protein complexes of bioenergetic membranes are structured to accomplish their biological functions. The goal of this project is to determine how pigment-protein complexes are able to convert absorbed light energy into electrochemical energy. The specific approach unique to this project involves the reconstitution of the structural subunit of the core light- harvesting complex of photosynthetic bacteria, and the reassociation of this subunit to form the in vivo complex. This is made possible by methodology developed in our laboratory and represents the first example of reconstitution of a chlorophy11- or of the few examples of such reconstitution of any complex, integral-membrane-protein system. Structural analogs of the pigment and specifically modified protein will be used to map out binding requirements. The protein consists of two small polypeptides several different bacteria. Reconstitution experiments will utilize selectively mixed polypeptides (an alpha polypeptide from one bacterium and a beta polypeptide from another) as a first step to learn some of the amino acid residues that are important to binding and pigment properties. Groups that have been implicated in providing ligands to coordinate to the Mg atom of bacteriochlorophy11 or to be involved in hydrogen bonding to the oxygen atoms of the molecule will be tested by chemical modification, site-directed mutagenesis, and/or chemical synthesis. Characterization of the reconstituted complexes will be by absorbance, circular dichroism, fluorescence, resonance Raman, epr, and high resolution nmr spectroscopies. It is hoped that by these combined methods it will be possible to unambiguously identify the binding sites of bacteriochlorophy11, and perhaps provide an overall 3-dimensional description. The specifically modified light-harvesting complexes will also be reassociated with the reaction center to re-form the photoreceptor complex (PRC) and probe the factors responsible for their specific association. Finally, the methodology developed to reconstitute the light- harvesting complexes will be extended in a systematic way to achieve reconstitution of the reaction center, as well. It is expected that the methodology being developed will also be applicable to reconstitution of other electron and proton transport clusters such as the bcl complex and cytochrome oxidase.
