Solid state 13C and 15N NMR investigations of the N intermediate of bacteriorhodopsin

Previous solid state 13C NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration and the protonation state of the retinal-lysine Schiff base (SB) linkage from the [13-13C]-retinal, [14-13C]retinal, and [epsilon-13C]lysine-216 chemical shifts in the bR555, bR568, and M412 states. Here we determine the C = N configuration and the protonation state of the N photointermediate that is cryotrapped along with the M photointermediate at high salt concentrations (0.1 M NaCl) and high pH (10.0). We obtained 13C and 15N SSN MR spectra of [epsilon-15N]lysine bR and [12-13C]- and [13-13C]retinal bR for samples illuminated under the above conditions. Two species are observed, both of which decay to bR568 upon warming. One species has chemical shifts identical to those obtained previously for M thermally trapped in guanidine.HCl at high pH (Smith et al., 1989a; Farrar et al., 1993). In the other species, the [epsilon-15N]lysine and 13-13C chemical shifts indicate that the SB is protonated, the 12-13C shift indicates a 13 = 14 cis configuration, and the previously published [14-13C]- and [epsilon-13C]lysine shifts indicate a C = N anti configuration. These results are consistent with other studies of the N photointermediate.