UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON
The long-term goal is to elucidate structure/function relationships in the microbial sensory rhodopsins, a family of visual pigment-like heptahelical photoreceptors, to elucidate principles of membrane receptor photoactivation and receptor interactions with membrane-bound and cytoplasmic transducers during signal transduction. Sensory rhodopsins I and II (SRI and SRII) are phototaxis receptors in haloarchaea, each of which couples to its cognate membrane-embedded transducer protein (Htrl and Htrll) to control a phosphorylation cascade that modulates the cell's motility. The SR receptors show close mechanistic similarities in their activation process to higher animal visual pigments, and their presence in a microorganism makes them more amenable to classical and molecular genetic analysis, combined with biochemical and biophysical approaches. In the current grant period the investigators have determined the dark structure of SRII at atomic resolution from two-dimensional (2-D) lipid bilayer crystals and three-dimensional (3-D) cubic lipid phase crystals by cryoelectron image analysis and x-ray diffraction. The applicants plan to use the crystals to obtain atomic resolution structures of SRII photocycle intermediates implicated in signal transfer to its transducer by applying freeze-trapping methods, as well as by crystallography of constitutively activated mutant forms of SRII bound to transducer fragments active in signal transfer. The structural information obtained will complement random mutagenesis, mutant selection, and functional analyses to identify residues critical in receptor activation and transducer signal relay, and will guide site-specific mutagenesis and time-resolved spectroscopy (ultraviolet (UV)-visible and FTIR spectroscopy) to monitor chemical events during the photosignal transduction process. Work over the past three years has revealed a previously unsuspected widespread family of photoactive proteins closely related to the haloarchaeal sensory rhodopsins. Organisms containing these proteins inhabit such diverse environments as soil, freshwater, salt-flats and ocean waters, and human and plant tissues as pathogens (e.g., the fungi Cryptococcus neoformans and Cryphonectria parasitica), and they comprise a broad phylogenetic range, including haloarchaea, proteobacteria, cyanobacteria, and eucaryotic microbes (fungi and algae). Experiments are designed to elucidate structure/function of selected members of the microbial rhodopsin family to test and expand the "unified mechanism" model for heptahelical protein photosignaling and transport put forth based on the analysis of the haloarchaeal proteins.