Nuclear magnetic resonance study of the schiff base in bacteriorhodopsin: Counterion effects on the ¹⁵N shift anisotropy

High-resolution, solid-state ¹⁵N NMR has been used to study the chemical shift anisotropies of the Schiff bases in bacteriorhodopsin (bR) and in an extensive series of model compounds. Using slow-spinning techniques, we are able to obtain sufficient rotational sideband intensity to determine the full ¹⁵N chemical shift anisotropy for the Schiff base nitrogen in bR₅₄₈ and bR₅₆₈. Comparisons are made between all-trans-bR₅₆₈ and N-all-trans-retinylidem butylimine salts with halide, phenolate, and carboxylate counterions. It is argued that for the model compounds the variation in ¹⁵N chemical shift reflects the variation in (hydrogen) bond strength with the various counterions. The results suggest that carboxylates and tyrosinates may form hydrogen bonds of comparable strength in a hydrophobic environment. Thus, the hydrogen bonding strength of a counterion depends on factors that are not completely reflected in the solution pK_a of its conjugate acid. For the model compounds, the two most downfield principal values of the ¹⁵N chemical shift tensor, δ₂₂ and δ–₃₃, vary dramatically with different counterions, whereas δ₁₁ remains essentially unaffected. In addition, there exists a linear correlation between δ₂₂ and δ₃₃, which suggests that a single mechanism is responsible for the variation in chemical shifts present in all three classes of model compounds. The data for bR₅₆₈ follow this trend, but the isotropic shift is 11 ppm further upfield than any of the model compounds. This extreme value suggests an unusually weak hydrogen bond in the protein.