The long term objective of this project is to use tools of molecular biology and biochemistry to understand the roles of various genes and their products in the regulation of function of a pigment - protein complex, Photosystem EL A more immediate objective is to understand the functional role of a number of very small proteins (< 10 kDa) in this multiprotein complex Our experimental organism is a unicellular, transformable cyanobacterium, Synechocystis 6803. In the past, we have used 'reverse genetics' approaches to create specific targeted mutations in a number of genes that encode various PSII proteins of Synechocystis 6803. We now plan to use similar approaches to create site directed mutants in genes encoding small proteins of this protein complex The PSII complex has structural as well as functional similarities with a reaction center, the first membrane bound multiprotein complex whose Xray crystal data have become available. However, the reaction center complex does not contain any small protein component. Interestingly, such small proteins are ubiquitous components of many membrane protein complexes, e.g., cytochrome oxidase. The structural and/or functional roles of such small proteins are, however, not clearly understood. e proposed studies in this project will thus, broaden our general understanding of the function of such small proteins in all protein-complexes of similar kind. These studies will also refine techniques and concepts that may be employed in future work on membrane protein complexes that are biomedically important. A combination of biochemical, genetic and recombinant DNA techniques will be used to study the effects various mutations in the site directed mutants created during this proposal period. In addition, we will to isolate randomly mutagenized PSII deficient mutants. Of particular interest will be the use of a transposon element, Tn5tac 1, to create conditional (IPTG, a lactose analog, -dependent) mutants that impaired in their PSII activities. Use of such versatile genetic tools and the facile transformation system Synechocystis 6803 will help us in identifying novel structural and, more important, regulatory loci that influence the biogenesis, stability, as well as function, of the PSII complex.
