Three proteins in the Halobacterium halobium phototaxis system have been identified: The ~25 kDa retinal-containing receptors sensory rhodopsins I and II (SR-I and SR-II), and a carboxylmethylated protein of 94 kDa Mr (MPP-I, methyl-accepting photoaxis protein I) involved in SR-I signalling. SR-I exists in two photoactive forms, one serving as an attractant receptor for orange-red light (lambdamax 587 nm), and the second as a repellent receptor for UV-blue light (lambdamax 373 nm). SR-II provides additional repellent sensitivity in the blue-green (lambda 487 nm). Photoisomerization of retinal in the SR-I or SR-II chromoprotein triggers a sequence of thermal steps returning the molecule to its original state. This cyclic process (or photocycle) produces receptor conformations (signalling states) which activate post-receptor components to modulate the flagellar motors. The signalling states of SR-I and SR-II are being identified by incorporating retinal analogs which alter lifetimes of photocycle intermediates, and measuring the photoaxis signalling efficiency of the analog receptors by computer-assisted motion analysis of swimming cells. SR-I, the better characterized of the two receptors, is similar in structure and in its retinal/apoprotein interactions to the light-driven proton pump bacteriorhodopsin (BR), yet unlike BR, SR-I is not active in ion translocation. To identify molecular processes during the SR-I photocycle which are essential for signalling, a synthetic SR-I chromoprotein gene (sopI) designed for casette mutagenesis will be site- specifically mutated and expressed in H. halobium by plasmid transformation. Initially a set of 12 residues implicated in photosignal transduction will be modified. The mutant receptors will be analyzed for spectroscopic properties in native membranes and for phototaxis signalling by analysis of cell swimming behavior. Our working hypothesis is that activated SR-I interacts with MPP-I, which relays the signal to the flagellar motor. SR-I/MPP-I protein/protein interaction will be tested and the stoichiometric relationship of MPP-I to SR-I determined. Sequences of proteolytic fragments of MPP-I will be used to design oligonucleotides probes for cloning. The gene-derived sequence will aid in the characterization of the MPP-I protein for the presence of membrane spanning segments, intracellular and extracellular domains, and the number and location of methylation sites.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027750-13
Application #
3275003
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-04-01
Project End
1995-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
13
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
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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
Govorunova, Elena G; Sineshchekov, Oleg A; Spudich, John L (2016) Proteomonas sulcata ACR1: A Fast Anion Channelrhodopsin. Photochem Photobiol 92:257-263
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

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