USE OF MUTANTS TO STUDY CYANOBACTERIAL MEMBRANE PROTEINS

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The long-term objective of this project is to develop and utilize genetic tools to study the biogenesis and architecture of membrane- bound protein complexes in cyanobacteria. The subject of the immediate investigation is the photosynthetic membrane and in particular, two integral protein complexes, Photosystem I (PSI) and Photosystem (PSII). The complex has structural as well as functional similarities with a bacterial reaction center, the first membrane protein complex whose xray crystal data have become available. This allows one to hypothesize about putative roles of different protein domains in PSII and use genetic approaches to examine these hypotheses. The unicellular, transformable cyanobacterium, Synechocystis 6803 will be used in this project for genetic analysis of protein-protein as well as protein-cofactor interactions in PSII. The PSI complex has also been crystallized in the past year and structural data are expected to be available soon. However, none of the already characterized cyanobacterial systems (including Synechocystis) can be used for genetic analysis of PSI. The proposed studies in this project will provide, for the first time, genetic tools to study the PSI complex in a prokaryotic organism, Nostoc 29150. Understanding the structure and assembly of these two integral membrane-bound protein complexes will be an important contribution to the general knowledge about all protein- complexes of similar kind. These studies will also refine techniques and concepts that will be employed in future work on membrane-protein complexes that are biomedically important. A combination of biochemical, genetic, biophysical and recombinant DNA techniques will be used to study the regulation of formation as well as the architecture of the PSI and PSII complexes. The immediate objectives in this proposal will be: 1) mutagenesis of the psbA gene, encoding a rapidly turned over 'D1' protein of the PSII complex in Synechocystis 6803; 2) cloning and mutagenesis of PSI genes from Nostoc 29150. The resultant mutants will then be analyzed to examine (a) whether mutation in one protein in a complex allows the rest of the complex to be assembled in the membrane and (b) which functional activities of a complex are inhibited by a specific mutation in a protein. We shall also perform random mutagenesis experiments to identify new structural and regulatory loci that play important roles in these protein complexes. Submembrane preparations will be developed for physicochemical analyses of these mutants.