Removal of the 9-methyl group of retinal inhibits signal transduction in the visual process. A Fourier transform infrared and biochemical investigation.
The photoreaction of opsin regenerated with 9-demethylretinal has been investigated by UV-vis spectroscopy, flash photolysis experiments, and Fourier transform infrared difference spectroscopy. In addition, the capability of the illuminated pigment to activate the retinal G-protein has been tested. The photoproduct, which can be stabilized at 77 K, resembles more the lumirhodopsin species, and only minor further changes occur upon warming the sample to 170 K (stabilizing lumirhodopsin). UV-vis spectroscopy reveals no further changes at 240 K (stabilizing metarhodopsin I), but infrared difference spectroscopy shows that the protein as well as the chromophore undergoes further molecular changes which are, however, different from those observed for unmodified metarhodopsin I. UV-vis spectroscopy, flash photolysis experiments, and infrared difference spectroscopy demonstrate that an intermediate different from metarhodopsin II is produced at room temperature, of which the Schiff base is still protonated. The illuminated pigment was able to activate G-protein, as assayed by monitoring the exchange of GDP for GTP gamma S in purified G-protein, only to a very limited extent (approximately 8% as compared to rhodopsin). The results are interpreted in terms of a specific steric interaction of the 9-methyl group of the retinal in rhodopsin with the protein, which is required to initiate the molecular changes necessary for G-protein activation. The residual activation suggests a conformer of the photolyzed pigment which mimics metarhodopsin II to a very limited extent.
Ganter
U M
UM
Institut für Biophysik und Strahlenbiologie, Albert-Ludwig-Universität Freiburg, FRG.
Schmid
E D
ED
Perez-Sala
D
D
Rando
R R
RR
Siebert
F
F
D000818:Animals; D005583:Fourier Analysis; D019204:GTP-Binding Proteins / Q000528:radiation effects; D020962:Heterotrimeric GTP-Binding Proteins; D066298:In Vitro Techniques; D015394:Molecular Structure; D010782:Photolysis; D012172:Retinaldehyde / Q000528:radiation effects; D012176:Retinoids / Q000528:radiation effects; D012243:Rhodopsin / Q000528:radiation effects; D015398:Signal Transduction / Q000528:radiation effects; D013055:Spectrophotometry, Infrared; D015293:Transducin; D014785:Vision, Ocular
C034809:GNAT1 protein, human; D012176:Retinoids; D012243:Rhodopsin; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D015293:Transducin; D012172:Retinaldehyde