Magic angle spinning NMR studies on the metarhodopsin II intermediate of bovine rhodopsin: evidence for an unprotonated Schiff base.
Magic angle spinning (MAS)13C-NMR spectra of the metarhodopsin II intermediate have been obtained using bovine rhodopsin regenerated with retinal 13C-labeled at the C-13 and C-15 positions to investigate the protonation state of the retinal Schiff base linkage. The 13C-labeled rhodopsin was reconstituted into 1,2-dipalmitoleoylphosphatidylcholine bilayers to increase the amount of meta II trapped at low temperature. Both the 13C-15 (159.2 ppm) and 13C-13 (144.0 ppm) isotropic chemical shifts are characteristic of an unprotonated Schiff base, while the 13C-15 shift is significantly different from that of retinal (191 ppm) or a tetrahedral carbinolamine group (70-90 ppm) previously proposed as an intermediate in the hydrolysis of the Schiff base at the meta II stage. This rules out the possibility that meta II non-covalently binds retinal or is a carbinolamine intermediate and provides convincing evidence that Schiff base deprotonation occurs in the meta I-meta II transition, an event that is likely to be important in triggering the activation of transducin.
Smith
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SO
Department of Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
de Groot
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H
Gebhard
R
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Lugtenburg
J
J
D000818:Animals; D002417:Cattle; D009682:Magnetic Resonance Spectroscopy; D010777:Photochemistry; D011522:Protons; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000528:radiation effects; D012545:Schiff Bases / Q000737:chemistry
D011522:Protons; D012545:Schiff Bases; C013110:metarhodopsins; D012243:Rhodopsin