Characterization of rhodopsin-transducin interaction: a mutant rhodopsin photoproduct with a protonated Schiff base activates transducin.
Rhodopsin, a G protein-coupled seven-transmembrane helix receptor, contains an 11-cis-retinal chromophore covalently linked to opsin apoprotein by a protonated Schiff base. Photoisomerization of the chromophore followed by Schiff base deprotonation forms metarhodopsin II (MII, lambda max = 380 nm), the active state (R*) that catalyzes guanine nucleotide exchange in transducin, the G protein of the photoreceptor cell. Schiff base deprotonation is required for R* formation. The Schiff base positive charge in rhodopsin is stabilized by a carboxylic acid counterion, Glu113. The position of the carboxylate counterion was moved by one helix turn to position 117 by site-specific mutagenesis. Photolysis of the mutant pigment E113A/A117E (lambda max = 491 nm) resulted in a mixture of two photoproducts: (1) an MII-like form with an unprotonated Schiff base (lambda max = 382 nm) favored at alkaline pH; and (2) a photoproduct with a protonated Schiff base (lambda max = 474 nm), spectroscopically similar to metarhodopsin I, favored at acidic pH. Here, we have studied the interactions between the mutant E113A/A117E photoproducts and transducin in detail. Transducin slowed down thermal conversion of the 474 nm form to the 382 nm form by stabilizing the 474 nm photoproduct. This effect was maximal at the pH optimum of transducin activation by the mutant R* and was abolished in the presence of GTP gamma S. In addition, the amount of the 474 nm species correlated with transducin activation rates during the thermal conversion of the photoproduct mixture. Thus, the 474 nm photoproduct of the mutant pigment, which contained a protonated Schiff base, activated transducin.(ABSTRACT TRUNCATED AT 250 WORDS)
Zvyaga
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Howard Hughes Medical Institute, Laboratory of Molecular Biology and Biochemistry, Rockefeller University, New York, New York 10021.
Fahmy
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Sakmar
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D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011522:Protons; D012243:Rhodopsin / Q000235:genetics* / Q000378:metabolism* / Q000528:radiation effects; D012545:Schiff Bases; D015398:Signal Transduction; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism* / Q000528:radiation effects
D011522:Protons; D012545:Schiff Bases; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin; D015293:Transducin