title: Photostimulation of a sensory rhodopsin II/HtrII/Tsr fusion chimera activates CheA-autophosphorylation and CheY-phosphotransfer in vitro.
abstract: A chimeric fusion protein consisting of Natronomonas pharaonis sensory rhodopsin II (SRII), fused by a flexible linker to the two transmembrane helices of its cognate transducer protein, HtrII, followed by the HtrII membrane-proximal cytoplasmic fragment joined to the cytoplasmic domains of the Escherichia coli chemotaxis receptor Tsr, was expressed in E. coli. Purified fusion chimera protein reconstituted in liposomes binds to E. coli CheA kinase in the presence of the coupling protein CheW, and activates CheA autophosphorylation activity. CheA kinase activity is stimulated by photoexcitation of the SRII domain of the fusion protein, as shown by the wavelength-dependence of photostimulated phosphotransfer to the E. coli flagellar motor response regulator CheY in the purified in vitro liposomal system. Further confirming the fidelity of the in vitro system, increased and decreased levels of CheA activation in vitro result from overmethylated and undermethylated fusion protein purified from methylesterase and methyltransferase-deficient E. coli, respectively. Photoexcitation of the undermethylated fusion protein resulted in a 3-fold increase in phosphotransfer over that of the dark state. The results directly demonstrate the coupling of SRII photoactivated states to histidine kinase activity, previously predicted on the basis of sequence homologies of the haloarchaeal phototaxis system components to those of E. coli chemotaxis. The fusion chimera provides the first tool for in vitro measurement of photosignaling activity of SRII-HtrII molecular complexes.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA.
authors.forename: Vishwa D
authors.initials: VD
authors.lastname: Trivedi
authors.forename: John L
authors.initials: JL
authors.lastname: Spudich
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002338:Carotenoids / Q000737:chemistry* / Q000235:genetics; D002633:Chemotaxis / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025602:Halorhodopsins; D000071677:Histidine Kinase; D040542:Mechanotransduction, Cellular / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D008745:Methylation; D019633:Natronobacterium / Q000737:chemistry / Q000235:genetics; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010777:Photochemistry; D011494:Protein Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D025603:Sensory Rhodopsins
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D025602:Halorhodopsins; C434451:HtrII protein, Natronobacterium pharaonis; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D025603:Sensory Rhodopsins; C057635:Tsr protein, Bacteria; C000607606:cheY protein, E coli; C061508:sensory rhodopsin II protein, archaeal; C000607604:tsr protein, E coli; D002338:Carotenoids; D011494:Protein Kinases; D000071677:Histidine Kinase; C000607608:cheA protein, E coli