Bacteriorhodopsin

Bacteriorhodopsin is a small (26 kDa) integral membrane protein, the prototype of seven-helical G-protein–linked receptors, that upon illumination transports protons across the membrane. It forms extended two-dimensional hexagonal arrays in the cytoplasmic membrane of halobacteria. Its transmembrane a-helices surround the prosthetic group retinal, which is linked via a Schiff Base to Lys216 near the middle of helix G and lies at a small angle to the membrane plane. Photoisomerization of the retinal from all-trans to 13-cis sets off a sequence of thermal reactions (the “photocycle”) in which the interaction of the retinal and the protein causes proton transfers between various donor and acceptor groups. Together, these transfers result in the complete translocation of a proton from the cytoplasmic to the extracellular surface, thus generating a transmembrane electrochemical gradient for protons. The proton gradient is utilized in the way usual in bacteria for the synthesis of ATP, the uptake of nutrients (amino acids) and K+, and the transport of Na+ out of the cells.

Bacteriorhodopsin forms trimers that assemble in the two-dimensional hexagonal array that constitutes the patches termed “purple membrane.” The purple membrane contains only bacteriorhodopsin and lipids, and the regular crystalline lattice made it possible to determine its structure by cryo-electron crystallography at 7 Å (1) and then 3 Å (2) resolution. The protein was also crystallized from a lipid cubic phase, and its structure has now been determined by X-ray crystallography at 2.5 Å resolution (3). The protein consists of seven transmembranous a-helices, with short interhelical loops and short N and C termini. Three of the helices, B, C, and D, are normal to the plane of the membrane and the other four, A, E, F, and G, are inclined at various small angles to the perpendicular (Fig. 1). The retinal is bound to the  -amino group of Lys216, forming a protonated Schiff base near the middle of helix G, and its polyene chain lies at about 23° from the membrane plane. Thus the Schiff base divides the protein into extracellular and cytoplasmic halves.

Figure 1. Structure of bacteriorhodopsin (1), and the pathway of proton transport. The seven transmembranous a-helices are shown, along with only the all-trans retinal and the most important residues. The curved arrows identify the proton transfers that occur at different times in the photocycle (see text) and add up to the complete transport of a proton from the cytoplasmic (upper) to the extracellular surface of the membrane.

The trajectory of the transported proton from one surface to the other is through proton-conductive “half-channels” through each of the two halves of the molecule. Identification of the residues that participate in the two half-channels has been the objective of much research in the past few years. The extracellular half-channel is quite complex and contains polar and hydrogen-bonding residues. Many of them play roles in the release of protons to the extracellular surface. Asp85 and Asp212 are anionic residues located near the Schiff base. A possible pathway for protons leads from Asp85, via Arg82, Glu204, and Glu194, to the extracellular surface and is consistent with functional studies. Another plausible pathway is through Glu9 or Glu74, but the results of mutagenesis indicate that these two residues are dispensable. The cytoplasmic half-channel is simpler. It contains mostly hydrophobic residues between the retinal and Asp96, which is the proton donor in the reprotonation of the Schiff base. Thr46 is close enough to interact with Asp96, and mutagenesis confirms its significant role in the proton donor and acceptor function of Asp96. Acidic residues at the cytoplasmic aqueous interface include residues 36, 38, 102, and 104. It has been suggested that they form a “funnel” at the surface that directs protons into the cytoplasmic half-channel (2). Although immobilization of the protein in the purple membrane is responsible for the well-known, extraordinary thermal and photostability of bacteriorhodopsin, neither the trimeric arrangement of the monomers nor the rigid lattice structure have functional roles in the transport activity (4). Thus, availability of the structure of bacteriorhodopsin, even at the current limited resolution, has generated considerable insights into the proton transport mechanism and has provided the framework for mechanistically interpreting a wealth of spectroscopic information.

The chromophore with all-trans, 15-anti retinal has a broad absorption band with a maximum at 568 nm. This is considerably red-shifted from that of retinal in isolation (380 nm) or a retinal analog with a protonated Schiff base (440 nm). This red-shift results primarily from the diffuse counterion to the charged Schiff base, which comprises principally Asp85 with a contribution from Asp212, which is several angstroms removed and providing only partial compensation of the charge (5). The all-trans chromophore exists in thermal equilibrium with the 13-cis, 15-syn configuration, which absorbs at 555 nm. Sustained illumination converts the retinal to 100% all-trans, 15-anti, known as “light-adaptation” (6). The photoreaction of this isomer is normally active in transport.

 The photocycle of the all-trans chromophore is described by the intermediate states J, K, L, M, N, and O, their substates, and the sequence of their interconversions (Fig. 2). Each intermediate is characterized by a distinct absorption maximum in the visible wavelength region, numerous vibrational bands of the retinal in the infrared and Raman region and of the protein in the infrared region. The kinetics of the photocycle describe the sequence and the energetics of the chemical reactions that translocate protons across the membrane. In the K intermediate, which arises on the nanosecond timescale after decay of the excited state, the retinal assumes a twisted 13-cis, 15-anti configuration, as indicated by high-amplitude hydrogen-out-of-plane vibrations. Its absorption maximum is red-shifted by 30 to 40 nm from the initial state. It is converted in about one microsecond into the L state, which absorbs at 540 to 550 nm. In L, the polyene of the retinal is more relaxed, but other changes begin to appear in the protein in both the extracellular and cytoplasmic regions (7). The Schiff base forms stronger hydrogen bonds that are correlated with structural changes of bound water detected in the infrared wavelength region. One of these water molecules is bound to the Schiff base, Asp85, and Asp212 and may play a role in the pK_a shifts that result in proton transfer from the Schiff base to Asp85. The hydrogen bonding of two other water molecules near Asp96 are also affected in L, suggesting that the structural changes near the Schiff base are transmitted all the way across the protein to the cytoplasmic region.

Figure 2. The photocycle of bacteriorhodopsin. The intermediate states are shown, and the isomeric configuration of the retinal is indicated. The Schiff base is protonated in all but the M states.

The M state is formed by transfer of a proton from the retinal Schiff base to Asp85 (8). It has a strongly blue-shifted absorption maximum at 410 nm. The kinetics of this conversion, measured at visible and infrared wavelengths, suggest that L is in equilibrium with an early M state, M1, the mixture of L and M1 decay together to form the late M state, M2 (9-11). At pH > 6 this occurs in a unidirectional reaction, and thus L disappears as M2 is formed. At pH < 6, however, the M1 → M2 reaction is not unidirectional, and both L and M1 remain present and coexist with M2 because at the higher pH a proton dissociates from a site in the extracellular region that interacts with Asp85. This site is either Glu204 or depends on the carboxyl group of Glu204. Its proton is passed to Glu194 and is released from there to the extracellular surface. The anomalous titration properties of Asp85 indicate that the nature of the interaction between the proton affinities of this aspartate residue and the proton release site is such that either may be protonated but not both (12, 13). When Asp85 becomes protonated by the Schiff base, the pK_a of the proton release group is lowered, and the proton dissociates. When this proton is released to the bulk solution at a pH greater than the pK_a for the release, the pKa of Asp85, in turn, is driven higher, and deprotonation of the Schiff base becomes complete. This prevents reprotonation of the Schiff base from the extracellular direction.

 Reprotonation of the Schiff base is from the cytoplasmic direction by Asp96 (8, 14), which produces the N intermediate that absorbs near 560 nm but with a lower extinction than the initial state. Large-scale protein conformational changes in N are evident from a pair of negative and positive difference features in the infrared spectrum that originate from a shift of the amide I band (15). Electron and X-ray diffraction studies of the M and the N states, measured either at various times after flash illumination and freezing or in a photostationary state at ambient temperature, indicate considerable changes of conformation at the cytoplasmic surface. The most conspicuous of these is an outward tilt of the cytoplasmic end of helix F (16). Its occurrence in the M intermediate may be transitory during the M → N conversion of the unperturbed wild-type photocycle, but this feature is clearly observable when M is stabilized in the wild-type or in mutant forms of the protein. The movement of helix F as a rigid body is confirmed by distance measurements using pairs of spin labels (17). The effects of osmotic agents, humidity, and in-plane cooperativity in the purple membrane lattice on the M → N reaction and on the protein conformation change suggest that the rationale of the helical tilt is to increase the hydration of the cytoplasmic region and thereby to decrease the pKa of Asp96. Thus, Asp96 becomes a proton donor to the Schiff base. The tilt of helix F is recovered during decay of the N state, presumably recovering the initial high pKa of Asp96 and causing its reprotonation from the cytoplasmic surface.

Reisomerization of the retinal to all-trans occurs in the N → O transition (18). This is made possible by the lowered barrier-to-bond rotation in the polyene chain upon protonation of the Schiff base. Residues that contact the chain near the 9-methyl and 13-methyl groups, such as Trp182 (19) and Leu93 (20), facilitate the reisomerization, probably through steric interactions that transmit residue displacements in the protein to the retinal and vice versa. The O state has a strongly red-shifted maximum at visible wavelengths, at least partly because Asp85 is still protonated. Consequently, the main component of the counterion to the protonated Schiff base is lacking. Large-amplitude hydrogen-out-of-plane vibrations indicate that, as in the K state, the retinal chain is twisted. These features disappear in the final O → BR reaction, which appears to be limited by the rate of proton transfer from Asp85 to the still unprotonated proton release site (21). As expected from the recovery of the low initial pKa of Asp85, this reaction is unidirectional under all conditions, and it ensures the full repopulation of the initial state and also the functioning of the proton pump against large transmembranous proton gradients.

 Bacteriorhodopsin is one of three types of similar retinal proteins in halobacterial membranes. Their functions are all based on the photoisomerization of all-trans retinal to 13-cis, 15-anti and the protein reactions that accompany the thermal reisomerization. Halorhodopsin is an inwardly-directed, light-driven chloride ion pump. It lacks Asp85 and Asp96, and the retinal Schiff base does not deprotonate during the photocycle (22). Sensory rhodopsins I and II are receptors for phototactic behavior (23). A profound similarity in the mechanisms of these proteins with different functions is indicated by the fact that their activities are interconvertible with minimal perturbations. Thus, the Asp85Thr mutant of bacteriorhodopsin binds chloride, exhibits a photocycle similar to that of halorhodopsin, and transports chloride from the extracellular to the cytoplasmic direction (24). Halorhodopsin, in turn, transports protons when the weak acid, azide, is added, by binding near the Schiff base and functioning as a proton acceptor (25). Sensory rhodopsin I transports protons like bacteriorhodopsin when the transducing protein that is normally tightly bound to it is genetically deleted (26, 27).

References

1.N. Grigorieff et al. (1996) J. Mol. Biol. 259, 393–421. 

2.Y. Kimura et al. (1997) Nature 389, 206–211. 

3.E. Pebay-Peyroula, G. Rummel, J. P. Rosenbusch, and E. M. Landau (1997) Science 277, 1676–1681.

4.N. A. Dencher and M. P. Heyn (1979) FEBS. Lett. 108, 307–310.  

5.K. Nakanishi et al. (1980) J. Am. Chem. Soc. 102, 7945–7947. 

6.G. S. Harbison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 1706–1709.

7.A. Maeda et al. (1997) J. Biochem. (Tokyo) 121, 399–406. 

8.M. S. Braiman et al. (1988) Biochemistry 27, 8516–8520. 

9.L. Zimányi et al. (1992) Biochemistry 31, 8535–8543. 

10.S. Dickopf and M. P. Heyn (1997) Biophys. J. 73, 3171–3181. 

11.B. Hessling, J. Herbst, R. Rammelsberg, and K. Gerwert (1997) Biophys. J. 73, 2071–2080. 

12.S. P. Balashov, E. S. Imasheva, R. Govindjee, and T. G. Ebrey (1996) Biophys. J. 70, 473–481. 

13.H. T. Richter, L. S. Brown, R. Needleman, and J. K. Lanyi (1996) Biochemistry 35, 4054–4062.

14.K. Gerwert, B. Hess, J. Soppa, and D. Oesterhelt (1989) Proc. Natl. Acad. Sci. USA 86, 4943–4947.

15.M. S. Braiman, O. Bousché, and K. J. Rothschild (1991) Proc. Natl. Acad. Sci. USA 88, 2388–2392.

16.S. Subramaniam, M. Gerstein, D. Oesterhelt, and R. Henderson (1993) EMBO J. 12, 1–8. 

17.T. E. Thorgeirsson et al. (1997) J. Mol. Biol. 273, 951–957. 

18.S. O. Smith et al. (1983) Biochemistry 22, 6141–6148. 

19.O. Weidlich et al. (1996) Biochemistry 35, 10807–10814. 

20.J. K. Delaney, G. Yahalom, M. Sheves, and S. Subramaniam (1997) Proc. Natl. Acad. Sci. USA 94, 5028-5033.

21.H. T. Richter et al. (1996) Biochemistry 35, 15461–15466. 

22.D. Oesterhelt (1995) Israel J. Chem. 35, 475–494. 

23.W. D. Hoff, K. H. Jung, and J. L. Spudich (1997) Annu. Rev. Biophys. Biomol. Struct. 26, 223–258.

24.J. Sasaki et al. (1995) Science 269, 73–75. 

25.G. Váró, L. S. Brown, R. Needleman, and J. K. Lanyi (1996) Biochemistry 35, 6604–6611. 

26.R. A. Bogomolni et al. (1994) Proc. Natl. Acad. Sci. USA 91, 10188–10192. 

27. U. Haupts, C. Haupts, and D. Oesterhelt (1995) Proc. Natl. Acad. Sci. USA 92, 3834–3838.