A photosensitive protein which resembles the visual pigments of invertebrates functions as a phototaxis receptor in Halobacterium halobium. This integral membrane protein, called """"""""slow-cycling"""""""" or sensory rhodopsin (SR), has been identified on the basis of its spectroscopic properties and its requirement for the vitamin A-derived chromophore, retinal. We will use these properties to purify the photoactive molecule and determine its subunit composition and primary structure. (3H)retinal-labeling experiments are designed to identify the chromophoric polypeptide. The amino acid sequence, especially in the region of the retinal-binding site, will be compared to the known sequences of eucaryotic sensory rhodopsins (visual pigments) and the ion pumps bacteriorhodopsin and halorhodopsin, also found in the H. halobium membrane. The bacteria exhibit wavelength sensitivity, being attracted to red and repelled by blue light. A color-discrimination mechanism based on SR photochemical reactions has recently been proposed. The key aspect is that SR exists in two spectrally distinct forms, one of which is a transient photoproduct of the other, and each of which undergoes photochemical reactions that control the cell's swimming behavior. Experiments are designed to test this model and to advance our understanding of the coupling of receptor reactions to the flagellar motor response. These include phototaxis action spectra and motility responses in various stimulus regimes and in strains with SR altered by mutation and by incorporation of retinal analogs into the SR apoprotein. Swimming behavior will be monitored by computer analysis of digitized time-resolved images (with the EV1000 Motion Analysis System), and receptor reactions by flash photolysis. Mutants in photoreception and the sensory signaling pathway will be isolated and characterized. These will be used in biochemical studies to investigate the nature of the SR signals. Possibilities to be explored are a cytoplasmic transducer protein for which SR changes its affinity during sensory signaling, cyclic nucleotide changes, and divalent cation release and uptake during SR photochemical reactions. The existence of a sensory rhodopsin in a bacterium provides excellent opportunities for experimental research on the molecular basis of sensory transduction. Our immediate objectives are to purify the molecule, define its structure, and explore its physiological and biochemical function.
| Govorunova, Elena G; Sineshchekov, Oleg A; Li, Hai et al. (2017) Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications. Annu Rev Biochem 86:845-872 |
| Yi, Adrian; Li, Hai; Mamaeva, Natalia et al. (2017) Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K. Biochemistry 56:2197-2208 |
| Sineshchekov, Oleg A; Govorunova, Elena G; Li, Hai et al. (2017) Bacteriorhodopsin-like channelrhodopsins: Alternative mechanism for control of cation conductance. Proc Natl Acad Sci U S A 114:E9512-E9519 |
| Govorunova, Elena G; Sineshchekov, Oleg A; Rodarte, Elsa M et al. (2017) The Expanding Family of Natural Anion Channelrhodopsins Reveals Large Variations in Kinetics, Conductance, and Spectral Sensitivity. Sci Rep 7:43358 |
| Yi, Adrian; Mamaeva, Natalia; Li, Hai et al. (2016) Resonance Raman Study of an Anion Channelrhodopsin: Effects of Mutations near the Retinylidene Schiff Base. Biochemistry 55:2371-80 |
| Li, Hai; Sineshchekov, Oleg A; Wu, Gang et al. (2016) In Vitro Activity of a Purified Natural Anion Channelrhodopsin. J Biol Chem 291:25319-25325 |
| Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L (2016) Structurally Distinct Cation Channelrhodopsins from Cryptophyte Algae. Biophys J 110:2302-2304 |
| Govorunova, Elena G; Cunha, Shane R; Sineshchekov, Oleg A et al. (2016) Anion channelrhodopsins for inhibitory cardiac optogenetics. Sci Rep 6:33530 |
| Sineshchekov, Oleg A; Li, Hai; Govorunova, Elena G et al. (2016) Photochemical reaction cycle transitions during anion channelrhodopsin gating. Proc Natl Acad Sci U S A 113:E1993-2000 |
| Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L (2016) Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin. Photochem Photobiol 92:257-263 |
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