Dark-adapted bacteriorhodopsin contains 13-cis,15-syn and all-trans,15-anti retinal Schiff bases

¹³C NMR spectra of lyophilized dark-adapted [14-¹³C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the ¹³C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called γ effect, which results from a steric interaction between the C-14 retinal proton and the protons on the ε CH₂ of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans,15-anti (trans or E) and 13-cis,15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bR₅₆₈ and bR₅₄₈. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with ¹³C shifts of the carbons adjacent to the C=N linkage in protonated ketimines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.