Signal reception and signal transduction constitute fundamental processes by which living cells perceive and ultimately react to external stimuli such as light, chemicals (nutrients, toxins, hormones), heat and mechanical signals. Most signal receptors reside in the cytoplasmic membrane that separates the inside of a cell from the external medium. Primary receptors for light (photoreceptors) are the basis of all forms of vision where the energy from absorbed single photons is used to bring about a conformational change in the chromophore, usually the isomerization of a double bond within picoseconds. On a slower timescale, the surrounding protein and water matrix responds to the conformational change of the chromophore and ultimately results in conformational and electrostatic changes that are sensed by a transducer molecule. Microbial rhodopsins are a family of photoactive, seven-transmembrane helix, retinylidene proteins found in phylogenetically diverse microorganisms, including haloarchaea, proteobacteria, cyanobacteria, fungi, and algae. Sensory rhodopsins I and II (SRI and SRII) in haloarchaea, Chlamydomonas rhodopsins CsoA and CsoB, and Anabaena rhodopsin, are photosensory receptors, spectrally tuned throughout the visible spectrum to relay information to the cell regarding the intensity and color of light in the environment. SRI and SRII are sensors for phototaxis in Hatobacterium salinarum and related halophilic archaea. SRI mediates attractant motility responses to green-orange wavelengths used by the ion pumps BR and HR, while SRII mediates blue-light avoidance responses. The SRI and SRII proteins are subunits of multicomponent signaling complexes and relay signals by protein-protein interaction to integral membrane transducer proteins (HtrI and HtrII, respectively) that control a cytoplasmic phosphorylation pathway that modulates the cell's motility apparatus. In order to elucidate the basis of spectral tuning of the photoreceptor sensory rhodopsin (SRII) and to obtain a detailed structure-based mechanism of how light-induced conformational changes trigger signaling in its cognate transducer (HtrII) we will 1. Determine the structure of the single-site mutant R72A, a residue recently implicated in spectral tuning. 2, Determine the crystal structure of the photoreceptor in the long-lived (O) signaling state and identify the main signaling components by comparing it to the recently solved ground state structure. 3, Compare the crystal structure of the 9-TM helical SRII/HtrlI fusion construct in the off-state with that of the photoactivated signaling state, In order to determine the atomic structure and the photoactivation mechanism of the recently identified Anabaena photoreceptor and its soluble putative transducer we will 4. Determine the crystal structure of the soluble putative transducer, a 14-kDa protein on the same operon as the photoreceptor with no sequence homology to any known protein. 5. Determine the crystal structure of the primary Anabaena photoreceptor in the ground and signaling state. 6. Determine the structure of the complex of the Anabaena photoreceptor with its putative transducer. ? ?
