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Solid-state 13C magic angle sample spinning NMR spectroscopy has been used to study the ionone ring portion of the chromophore of bacteriorhodopsin. Spectra were obtained from fully hydrated samples regenerated with retinals 13C labeled at positions C-5, C-6, C-7, C-8, and C-18 and from lyophilized samples regenerated with retinals labeled at C-9 and C-13. C-15-labeled samples were studied in both lyophilized and hydrated forms. Three independent NMR parameters (the downfield element of the C-5 chemical shift tensor, the C-8 isotropic chemical shift, and the C-18 longitudinal relaxation time) indicate that the chromophore has a 6-s-trans conformation in the protein, in contrast to the 6-s-cis conformation that is energetically favored for retinoids in solution. We also observe an additional 27 ppm downfield shift in the middle element of the C-5 shift tensor, which provides support for the existence of a negatively charged protein residue near C-5. Evidence for a positive charge near C-7, possibly the counterion for the negative charge, is also discussed. On the basis of these results, we present a new model for the retinal binding site, which has important implications for the mechanism of the "opsin shift" observed in bacteriorhodopsin.

ABSTRACT13C NMR spectra of Iyophilized 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 y effect, which results from a steric interaction between the C-14 retinal proton and the protons on the e 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 bRm and bR54. 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 ketinines. 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.Bacteriorhodopsin (bR), the single protein of the purple membrane (PM) of Halobacterium halobium (1), has been the subject of considerable experimental scrutiny for some time. Like rhodopsin (2), it contains as its chromophore the polyene aldehyde retinal, connected via a Schiff base linkage to the E-amino group of a lysine side chain (3). Although bR appears to resemble visual pigments (4) (14), the configuration of the C=-N Schiff base linkage in bR has not been considered. In addition, the interesting possibility that isomerization about this bond might occur during the photocycle has been largely overlooked. The inattention to this important question can be attributed in part to the absence of an experimental means to discriminate definitively between syn and anti isomers in bR. In a recent paper (15) we demonstrated that high-resolution solid-state 3C NMR is a potent means of establishing configuration about C=C bonds in bR. Here, we present evidence that it is equally effective in determining the C=N bond configuration and evinces that dark-adapted bR contains alltrans,15-anti and 13-cis,15-syn isomers in an approximately 4:6 ratio. The implications of this finding for both the structure of the chromophore and the bR photocycle will also be discussed. MATERIALS AND METHODS13C-14-labeled retinal was prepared by the method of ref. 16 and incorporated into white membrane as described (8, 15). The reconstituted PM was then lyophilized at 0.1 mm Hg (1 mm Hg = 133 Pa) and packed into a Kel-F rotor of the Andrew-Beams design (17). 13C magic-angle sample spinning (MASS) spectra were obtained at various spinning frequencies between 1.9 and 3.2 kHz, with a 13C frequency of 79.9 MHz. Typically, (,)1/21r) = 50 kHz was used for cross-polarization. Subsequently, the magnetization was sampled in the presence of 1H decoupling fields of 125 kHz. Usually, 15,000 transients were accumulated, with a recycle de...

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-retinylidene 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 pKa of its conjugate acid. For the model compounds, the two most downfield principal values of the 15N chemical shift tensor, sigma 22 and sigma 33, vary dramatically with different counterions, whereas sigma 11 remains essentially unaffected. In addition, there exists a linear correlation between sigma 22 and sigma 33, 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.

In reconstituted vesicles above the lipid phase transition temperature, bacteriorhodopsin (BR) undergoes rotational diffusion about an axis perpendicular to the plane of the bilayer [Cherry, R. J., Muller, U., & Schneider, G. (1977) FEBS Lett. 80, 465]. This diffusion narrows the 13C NMR powder line shape of the BR peptide carbonyls. In contrast, BR in native purple membrane is relatively immobile and exhibits a rigid-lattice powder line shape. By use of the principal values of the rigid-lattice chemical shift tensor and the motionally narrowed line shape from the reconstituted system, the range of Euler angles of the leucine peptide groups relative to the diffusion axis has been calculated. The experimentally observed line shape is inconsistent with those expected for structures which consist entirely of either alpha helix or beta sheet perpendicular to the membrane or beta sheet tilted at angles up to about 60 degrees from the membrane normal. However, for two more complex structural models, the predicted line shapes agree well with the experimental one. These are, first, a structure consisting entirely of alpha1 helices tilted at 20 degrees from the membrane normal and, second, a combination of 60% alpha II helix perpendicular to the membrane plane and 40% antiparallel beta sheet tilted at 10-20 degrees from the membrane normal. The results also indicate that the peptide backbone of bacteriorhodopsin in native purple membrane is extremely rigid even at 40 degrees. The experiments presented here demonstrate a new approach, using solid-state nuclear magnetic resonance (NMR) methods, for structural studies of transmembrane proteins in fluid membrane environments, either natural or reconstituted.(ABSTRACT TRUNCATED AT 250 WORDS)