RUI: Interaction of Domains on CP 43 With Components Required for Oxygen Evolution

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9982981Putnam-EvansThis project is designed to probe the structure and function of the chlorophyll-binding protein CP 43 using mutagenesis techniques. CP 43 is a protein component of the Photosystem II (PSII) complex of higher plants, green algae and cyanobacteria (blue-green algae). PSII uses light energy from the sun to drive the splitting of water molecules with concomitant release of molecular oxygen to the atmosphere. Virtually all of the oxygen present in our atmosphere arises from water oxidation by PSII. The CP 43 protein is embedded in the thylakoid membrane; however, it contains several extrinsic loop regions, including a large extrinsic loop E, which protrude from the membrane into the interior (lumen) of the thylakoid (the site of water oxidation). CP 43 is known to play a role in the light-harvesting process. However, accumulating data point to additional roles for CP 43 in the stable assembly of the PS II complex and in the water splitting (oxygen-evolving) process. The PI hypothesizes that domains on the lumenal surface of CP 43, and in particular the large extrinsic loop E, interact with components of the oxygen-evolving complex. The PI's laboratory is testing this hypothesis by using genetic engineering techniques to produce mutants in all the lumenal extrinsic loops. This is accomplished by introducing specific base pair changes in regions of the psbC gene encoding the lumenal loops of CP 43, and then introducing the altered genes back into our model organism, the cyanobacterium Synechocystis 6803. The organism then produces a mutant protein containing a single amino acid substitution in one of the extrinsic loops. To date the PI has focused on alteration of charged amino acid residues in the lumenal domains. Twenty-one mutants at twenty-two sites in the large extrinsic loop E of the CP 43 protein of Synechocystis 6803 have been constructed. Four of these mutants assemble PSII centers but are impaired in PSII activity. One additional mutant fails to assemble any functional centers and thus is devoid of PSII activity. Eleven other amino acids in lumenal loops are targeted for mutagenesis. A combination of physiological, biochemical and biophysical techniques will be employed to analyze and further characterize these mutants.