RUI: Mutagenesis Studies to Probe Function of the Large Extrinsic Loop of the CP-43 Apoprotein of Photosystem II

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9513795 Putnam-Evans This project describes the production and characterization of mutants containing random and site-directed lesions in the large extrinsic loop region of the Photosystem II (PSII) protein CP43. CP43 is an integral thylakoid protein and component of the photosynthetic apparatus of higher plants, algae and cyanobacteria. These mutants will be constructed in the cyanobacterium Synechocystis 6803, a widely used model system for higher plant photosynthesis. CP43 appears to be required for the stable assembly of the PSII complex, and also appears to play a role in photosynthetic oxygen evolution. The goal of this project is to determine the function of the large, lumenally-oriented extrinsic loop of CP43. Deletion of long segments of the loop results in mutants incapable of evolving oxygen. Therefore, it is probable that this region interacts directly with other PSII proteins and inorganic cofactors (manganese, chloride and calcium) that are necessary for oxygen evolution. In addition, CP43 functions as a chlorophyll-a antenna for PSII. The large extrinsic loop may thus interact with other PSII antenna proteins or proteins comprising the PSII reaction center. Mutations will be introduced into the psbC gene encoding CP43 by both random and oligonucleotide-mediated mutagenesis. Mutations will be re-introduced into Synechocystis 6803 along with a closely-linked antibiotic resistance marker. Putative mutants will be screened initially for antibiotic resistance and possible loss of photoautotrophic growth. This will be followed by an in-depth characterization of photosynthetic characteristics including measurements of steady state rates of oxygen evolution, electron transport capability, PSII polypeptide composition, manganese and calcium content and chloride requirement. %%% The Photosystem II (PSII~ complex forms part ofthe photosynthetic apparatus of higher plants, algae and cyanobacteria. This complex catalyzes the light-driven oxidation of water and the reduction of plastoquinon e. Oxygen is given off as a by-product of the PSII reaction. CP43 is one of the protein components of PSII and appears to be required for the stable assembly ofthe PSII complex. It also appears to play a role in both photosynthetic oxygen evolution and the harvesting of light energy. This membrane-bound protein is unusual in that it contains a large, hydrophilic loop which protrudes from the membrane in which is it embedded. Deletion of short stretches of amino acids in the large extrinsic loop result in mutants that cannot evolve oxygen. The goal of this project is to identify specific amino acids involved in the function of the large, lumenally-oriented extrinsic loop of CP43 to further test the hypothesis that this region is necessary for normal PSII activity. This will be accomplished by 1) introducing mutations into the gene encoding CP43 in vitro using the techniques of both random and site-specific mutagenesis, 2) exchanging these mutated genes for their normal counterparts in the blue-green algae Synechocystis 6803 and 3) evaluating the resulting mutant organisms for their ability to assemble a normal PSII complex, evolve oxygen and carry out electron transport. *** ??