Effects of substitutions D73E, D73N, D103N and V106M on signaling and pH titration of sensory rhodopsin II.
Several mutations in the repellent phototaxis receptor sensory rhodopsin II (SRII), in residues homologous to residues important in the related proton pump bacteriorhodopsin, were expressed in Pho81Wr-, a Halobacterium salinarum strain deficient in production of SRII and its transducer protein HtrII. The lack of production of SRII and HtrII is shown to be due to insertion of an ISH2 transposon into the promoter region upstream of the htrII-sopII gene pair. Near wild-type phototaxis responses are rescued in Pho81Wr- by expression of HtrII with D73E, D103N or V106M receptors. Partial responses are restored by the HtrII-D73N pair. From absorption spectroscopy of his-tag-purified receptor protein from mutants D73N and D73E we conclude that Asp73 is the primary counterion to the protonated Schiff base in SRII, like the corresponding Asp85 in bacteriorhodopsin. The absorption maximum of SRII (487 nm) is shifted to 514 nm in mutant D73N, a 1080 cm-1 shift identical to that caused by D85N in bacteriorhodopsin. Acid titration of SRII also induces the red shift with a pK of 3.0 in wild type. The absorption shift and the pK are nearly the same in V106M and D103N, but the pK is raised to 5.1 in D73E, confirming that Asp73 is the residue responsible for this spectral transition.
Zhu
J
J
Department of Microbiology & Molecular Genetics, University of Texas Medical School, Houston 77030, USA.
Spudich
E N
EN
Alam
M
M
Spudich
J L
JL
D019843:Archaeal Proteins; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002338:Carotenoids; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D006217:Halobacterium / Q000235:genetics / Q000378:metabolism; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010957:Plasmids; D025603:Sensory Rhodopsins; D015398:Signal Transduction; D013057:Spectrum Analysis
D019843:Archaeal Proteins; D004251:DNA Transposable Elements; D004274:DNA, Recombinant; D025602:Halorhodopsins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D002338:Carotenoids; D001436:Bacteriorhodopsins