MAGIC ANGLE SPINNING NMR STUDIES ON THE METARHODOPSIN II INTERMEDIATE OF BOVINE RHODOPSIN: EVIDENCE FOR AN UNPROTONATED SCHIFF BASE

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Abstract
Abstract— Magic angle spinning (MAS)W‐NMR spectra of the metarhodopsin II intermediate h been obtained using bovine rhodopsin regenerated with retinal 13C‐labeled at the C‐13 and C positions to investigate the protonation state of the retinal Schiff base linkage. The 13C‐labe rhodopsin was reconstituted into 1,2‐dipalmitoleoylphosphatidylcholine bilayers to increase the amo 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 “C‐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 the possibility that meta II non‐covalently binds retinal or is a carbinolamine intermediate and provi convincing evidence that Schiff base deprotonation occurs in the meta I‐meta II transition, an ev that is likely to be important in triggering the activation of transducin.