To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore-i.e., there must be at least two different M intermediates in the functional photocycle. We present here the first detection of multiple M intermediates in native wild-type bacteriorhodopsin by solid-state NMR. Illumination of light-adapted [zeta-15N-Lys]-bR at low temperatures shifts the 15N signal of the retinal Schiff base (SB) downfield by about 150 ppm, indicating a deprotonated chromophore. In 0.3 M Gdn-HCl at pH 10.0, two different M states are obtained, depending on the temperature during illumination. The M state routinely prepared at the lower temperature, Mo, decays to the newly observed M state, Mn, and the N intermediate, as the temperature is increased. Both relax to bR568 at 0 degreesC. A unique reaction sequence is derived: bR568-->Mo-->(Mn+N)-->bR568. Mo and Mn have similar chemical shifts at [12-13C]ret, [14-13C]ret, and [epsilon-13C]Lys216, indicating that Mn, like Mo, has a 13-cis and C=N anti chromophore. However, a small splitting in the [14-13C]ret signal of Mo reveals that it has at least two substates. The 7 ppm greater shielding of the SB nitrogen in Mn compared to Mo suggests an increase in basicity and/or hydrogen bonding. Probing the peptide backbone of the protein, via [1-13C]Val labeling, reveals a substantial structural change between Mo and Mn including the relaxation of perturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure and the increase in the pKa of the SB suggests that the demonstrated Mo-->Mn transition may function as the "reprotonation switch" required for vectorial proton transport.

15N solid-state NMR (SSNMR) spectra of guanidyl-15N-labeled bacteriorhodopsin (bR) show perturbation of an arginine residue upon deprotonation of the retinal Schiff base during the photocycle. At the epsilon position, an upfield shift of 4 ppm is observed while the eta nitrogens develop a pair of 'wing' peaks separated by 24 ppm. Proton-driven spin diffusion between the two 'wing' peaks indicates that they arise from a single Arg residue. An unusually asymmetric environment for this residue is indicated by comparison with guanidyl-15N chemical shifts in a series of arginine model compounds. The 'wing' peaks are tentatively assigned to Arg-82 on the basis of the SSNMR investigations of the alkaline and neutral dark-adapted forms of the D85N bacteriorhodopsin mutant. Another, less asymmetric pair of eta signals, that is not affected by Schiff base deprotonation or D85 mutation, is tentatively assigned to Arg-134. The results are discussed in relation to existing models of bR structure and function.

UV-visible and solid-state NMR studies of a series of6-s-trans protonated Schiffbases of retinal with aniline show that the bathochromic shift induced by weakening the imine counterion is sificandy greater in the 6-s-trans conformation than in the 6-s-cis conformation. Based on the observed magnitude of this coupling between the electronic effects of6-s isomerization and imine counterion strength in the mbdel compounds, the large opsin shift and unusual chemical shifts in light-adapted bacteriorhodopsin can be fully explained. These phenomena therefore do not require a negative point charge or polarizability effects in the chromophore binding pocket. The results are consistent with an effective center-to-center distance between the Schiff base and its counterion of about 4 A in light-adapted bacteriorhodopsin.Retinal pigments are found widely in nature, in organisms as evolutionarily distant as archae, algae, and mammals. In most cases, these integral membrane proteins are responsible for the first step of signal transduction. However, at least in archae, some retinal pigments function as energy transducers, by executing light-driven transport of ions. Certainly, for the visual pigments, and perhaps also for the ion pumps, it is important that the protein responds to light in the appropriate part of the spectrum. Since the chromophore in each known case is a protonated Schiff base (pSB) of retinal with a lysine residue, it is generally appreciated that the tuning of the chromophore must be due to the characteristics of the binding pocket in the protein. The shift of the wavelength of maximal visible absorbance (Amx), relative to the value of440 nm for the chloride of retinalbutylimine in methanol (1) (iv) The remaining identified mechanism for the opsin shift involves isomerization around the 6-s bond connecting the polyene chain to the ionone ring. Due to the methyl groups on the ionone ring, retinal in solution prefers the skewed 6-s-cis conformation. Isomerization to the planar 6-s-trans conformation results in a bathochromic shift due to extension ofthe polyene ir system to include the double bond in the ring. The magnitudes ofthe opsin shifts associated with these four mechanisms have been investigated using a variety of retinal analogs with appended charges (5, 6), disrupted ir systems (7, 8), and locked conformations (9), as well as variations of the pSB counterion (10-14) and solvents of varying polarizability (15)(16)(17).Where more than one opsin shift mechanism is at work, it has generally been assumed that the effects on the energy of excitation (or equivalently on .max = A 1 ) are additive.However, this has led to apparent inconsistencies. Here, we investigate the possibility of synergistic effects. We focus on the combination of the 6-s isomerization and the strength of the pSB counterion because (i) MATERIALS AND METHODSPreparation of pSBs. The pSBs were never exposed to white light. All syntheses and NMR experiments were conducted under red light or in darkness. To synthesize the pSBs,...