STATIC AND TIME-RESOLVED CRYSTALLOGRAPHIC STUDIES ON BACTERIOPHYTOCHROMES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phytochromes are a widespread family of photoreceptors that use linear tetrapyrrole molecules (bilins) as chromophores to sense light, primarily in the red and far-red region. They regulate a spectrum of physiological responses via reversible photoconversion between two thermo-stable, spectrally distinct forms, Pr and Pfr. First discovered in plants, phytochromes are also identified in cyanobacteria, purple bacteria, non-photosynthetic bacteria, and even fungi. Plant phytochromes are key regulators in many important developmental processes such as chlorophyll synthesis, seed germination, floral induction and shade avoidance, where their prokaryotic ancestors are found to be involved in key processes that control synthesis of photosystems and light harvest complexes, phototaxis, etc. The Z to E isomerization around C15-C16 double bond in linear tetrapyrrole bilin chromophore is believed to be the primary photoevent in the reversible photoconversion between Pr and Pfr forms, but little is known about molecular and mechanistic details of Pr/Pfr reversible photoconversion as well as intermediate structures in both forward and reverse processes. We will take advantage of both static and time-resolved X-ray crystallographic techniques to explore several bateriophytochrome systems, such as RpBphP2, RpBphP3, RpBphP5 from R. palustris, AtBphP2 from A. tumefaciens, and PaBphP from P. aeruginosa, with different domain combinations. Our specific aims are: 1. to reveal molecular basis of Pr/Pfr photoswitching; 2. to extract structural intermediates during Pr/Pfr photoconversion using both trapping and time-resolved techniques; 3. to understand how the light signal recieved by the N-terminal photosensory domains (PLD, GAF and PHY domains) is transduced into a chemical signal in the C-terminal effector histidine kinase; 4. to design chimeric proteins with defined and modulated photochemical properties using site-directed mutagensis and protein engineering.