GENETIC ANALYSIS OF THE PHOTOSYNTHETIC REACTION CENTER

点击翻译按钮获取中文摘要

Membrane-bound protein complexes are involved in many important health related processes including respiration, transport processes, transformation, sensory transduction, receptor binding, and signal transduction. the bacterial reaction center complex, which mediates a series of efficient electron transfer reactions resulting in the conversion of light into chemical energy, is the only membrane protein whose structure has been determined to atomic resolution. While the highest resolution structural data (2.3 angstroms) is available from the reaction centers of Rhodopseudomonas viridis, a system for the expression of genetically modified reaction centers in this organism is not available. We have been characterizing a heterotrophic strain of Rps. viridis which will be used to develop such a genetic system. the Rps. viridis reaction center system will provide the opportunity to test substrate recognition/binding requirements in membrane proteins and theories of electron transfer in biological systems, since the protein structure of modified complexes can be determined and then correlated with biochemical and spectroscopic studies of these same complexes. The puf operon of Rps. viridis (encoding the beta and alpha subunits of the light harvesting complex and the L subunit, M subunit, and cytochrome subunit of the reaction center) will be deleted from the Rps. viridis chromosome. A plasmid containing a copy of the puf operon will be used to complement this chromosomal deletion. Pigment-protein binding interactions throughout the reaction center will be modified by site- directed mutagenesis of the Rps. viridis structural genes, followed by crystallographic and spectroscopic characterization of the altered reaction center complexes. The insights provided by the Rps. viridis reaction center system will provide a basis for understanding of structure-function relationships in other membrane proteins where atomic resolution structural information is not available.