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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.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.

Solid-state 13C MAS NMR spectra were obtained for dark-adapted bacteriorhodopsin (bR) labeled with [4'-13C]Tyr. Difference spectra (labeled minus natural abundance) taken at pH values between 2 and 12, and temperatures between 20 and -90 degrees C, exhibit a single signal centered at 156 ppm, indicating that the 11 tyrosines are protonated over a wide pH range. However, at pH 13, a second line appears in the spectrum with an isotropic shift of 165 ppm. Comparisons with solution and solid-state spectra of model compounds suggest that this second line is due to the formation of tyrosinate. Integrated intensities indicate that about half of the tyrosines are deprotonated at pH 13. This result demonstrates that deprotonated tyrosines in a membrane protein are detectable with solid-state NMR and that neither the bR568 nor the bR555 form of bR present in the dark-adapted state contains a tyrosinate at pH values between 2 and 12. Deprotonation of a single tyrosine in bR568 would account for 3.6% of the total tyrosine signal, which would be detectable with the current signal-to-noise ratio. We observe a slight heterogeneity and subtle line-width changes in the tyrosine signal between pH 7 and pH 12, which we interpret to be due to protein environmental effects (such as changes in hydrogen bonding) rather than complete deprotonation of tyrosine residue(s).