Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.     

Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-13C]Val-labeled proteins.

We have recorded 13C cross-polarization-magic angle spinning and dipolar decoupled-magic angle spinning NMR spectra of [1-13C]Val-labeled wild-type bacteriorhodopsin (bR), and the V49A, V199A, T46V, T46V/V49A, D96N, and D85N mutants, in order to study conformational changes of the backbone caused by site-directed mutations along the extracellular surface and the cytoplasmic half channel. On the basis of spectral changes in the V49A and V199A mutants, and upon specific cleavage by chymotrypsin, we assigned the three well-resolved 13C signals observed at 172.93, 172.00, and 171. 11 ppm to [1-13C]Val 69, Val 49, and Val 199, respectively. The local conformations of the backbone at these residues are revealed by the conformation-dependent 13C chemical shifts. We find that at the ambient temperature of these measurements Val 69 is not in a beta-sheet, in spite of previous observations by electron microscopy and x-ray diffraction at cryogenic temperatures, but in a flexible turn structure that undergoes conformational fluctuation. Results with the T46V mutant suggest that there is a long-distance effect on backbone conformation between Thr 46 and Val 49. From the spectra of the D85N and E204Q mutants there also appears to be coupling between Val 49 and Asp 85 and between Asp 85 and Glu 204, respectively. In addition, the T2 measurement indicates conformational interaction between Asp 96 and extracellular surface. The protonation of Asp 85 in the photocycle therefore might induce changes in conformation or dynamics, or both, throughout the protein, from the extracellular surface to the side chain of Asp 96.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.     

Vibrational spectroscopy of bacteriorhodopsin mutants: chromophore isomerization perturbs tryptophan-86

Fourier transform infrared difference spectra have been obtained for the bR----K and bR----M photoreactions of bacteriorhodopsin mutants with Phe replacements for Trp residues 10, 12, 80, 86, 138, 182, and 189 and Cys replacements for Trp residues 137 and 138. None of the tryptophan mutations caused a significant shift in the retinylidene C = C or C-C stretching frequencies of the visible absorption maximum of the chromophore, it is concluded that none of the tryptophan residues are essential for forming a normal bR570 chromophore. However, a 742-cm-1 negative peak attributed previously to the perturbation of a tryptophan residue during the bR----K photoreaction was found to be absent in the bR----K and bR----M difference spectra of the Trp-86 mutant. On this basis, we conclude that the structure or environment of Trp-86 is altered during the bR----K photoreaction. All of the other Trp----Phe mutants exhibited this band, although its frequency was altered in the Trp-189----Phe mutant. In addition, the Trp-182----Phe mutant exhibited much reduced formation of normal photoproducts relative to the other mutants, as well as peaks indicative of the presence of additional chromophore conformations. A model of bR is discussed in which Trp-86, Trp-182, and Trp-189 form part of a retinal binding pocket. One likely function of these tryptophan groups is to provide the structural constraints needed to prevent chromophore photoisomerization other than at the C13 = C14 double bond.     

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.     

Structural investigation of the active site in bacteriorhodopsin: geometric constraints on the roles of Asp-85 and Asp-212 in the proton-pumping mechanism from solid state NMR

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.     

High-resolution solid state 13C NMR of bacteriorhodopsin: characterization of [4-13C]Asp resonances.

Solid state 13C nuclear magnetic resonance measurements of bacteriorhodopsin labeled with [4-13C]Asp show that resonances of single amino acids can be resolved. In order to assign and characterize the resonances of specific Asp residues, three different approaches were used. (1) Determination of the chemical shift anisotropy from side-band intensities provides information about the protonation state of Asp residues. (2) Relaxation studies and T1 filtering allow one to discriminate between resonances with different mobility. (3) A comparison of the spectra of light- and dark-adapted bacteriorhodopsin provides evidence for resonances from aspartic acid residues in close neighborhood of the chromophore. In agreement with other investigations, four resonances are assigned to internal residues. Two of them are protonated in the ground state up to pH 10 (Asp96 and Asp115). All other detected resonances, including Asp85 and Asp212, are due to deprotonated aspartic acid. Two lines due to the two internal deprotonated groups change upon dark and light adaptation, whereas the protonated Asp residues are unaffected.     

FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Bacteriorhodopsin (bR) is a light-driven proton pump whose function includes two key membrane-based processes, active transport and energy transduction. Despite extensive research on bR and other membrane proteins, these processes are not fully understood on the molecular level. In the past ten years, the introduction of Fourier transform infrared (FTIR) difference spectroscopy along with related techniques including time-resolved FTIR difference spectroscopy, polarized FTIR, and attenuated total reflection FTIR has provided a new approach for studying these processes. A key step has been the utilization of site-directed mutagenesis to assign bands in the FTIR difference spectrum to the vibrations of individual amino acid residues. On this basis, detailed information has been obtained about structural changes involving the retinylidene chromophore and protein during the bR photocycle. This includes a determination of the protonation state of the four membrane-embedded Asp residues, identification of specific structurally active amino acid residues, and the detection of protein secondary structural changes. This information is being used to develop an increasingly detailed picture of the bR proton pump mechanism.     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).     

The structure of bacteriorhodopsin at 3.0 A resolution based on electron crystallography: implication of the charge distribution

Electron crystallography has the potential to visualise the charge status of atoms. This is due to the significantly different scattering factors of neutral and ionised atoms for electrons in the low-resolution range (typically less than 5 A). In previous work, we observed two different types of densities around acidic residues in the experimental (|Fo|) map of bacteriorhodopsin (bR), a light-driven proton pump. We suggested that these might reflect different states of the acidic residues; namely, the protonated (neutral) and the deprotonated (negatively charged) state. To evaluate the observed charge more quantitatively, we refined the atomic model for bR and eight surrounding lipids using our electron crystallographic data set between 8.0 and 3.0 A resolution, where the charge effect is small. The refined model yielded an R-factor of 23.7% and a free R-factor of 33.0%. To evaluate the effect of charges on the density map, we calculated a difference (|Fo|-|Fc|) map including data of a resolution lower than 8.0 A resolution, where the charge effect is significant. We found strong peaks in the difference map mainly in the backbone region of the transmembrane helices. We interpreted these peaks to come from the polarisation of the polar groups in the main chain of the alpha-helices and we examined this by assuming a partial charge of 0.5 for the peptide carbonyl groups. The resulting R and free R-factors dropped from 0.250 and 0.341 to 0.246 and 0.336, respectively. Furthermore, we also observed some strong peaks around some side-chains, which could be assigned to positively charged atoms. Thus, we could show that Asp36 and Asp102 are likely to interact with cations nearby. In addition, peaks found around the acidic residues Glu74, Glu194 and Glu212 have different features and might represent positive charges on polarised water molecules or hydroxonium ions.     

Mechanism of light-dependent proton translocation by bacteriorhodopsin.

Site-specific mutagenesis has identified amino acids involved in bR proton transport. Biophysical studies of the mutants have elucidated the roles of two membrane-embedded residues: Asp-85 serves as the acceptor for the proton from the isomerized retinylidene Schiff base, and Asp-96 participates in reprotonation of this group. The functions of Arg-82, Leu-93, Asp-212, Tyr-185, and other residues that affect bR properties when substituted are not as well understood. Structural characterization of the mutant proteins will clarify the effects of substitutions at these positions. Current efforts in the field remain directed at understanding how retinal isomerization is coupled to proton transport. In particular, there has been more emphasis on determining the structures of bR and its photointermediates. Since well-ordered crystals of bR have not been obtained, continued electron diffraction studies of purple membrane offer the best opportunity for structure refinement. Other informative techniques include solid-state nuclear magnetic resonance of isotopically labeled bR (56) and electron paramagnetic resonance of bR tagged with nitroxide spin labels (2, 3, 13, 15). Site-directed mutagenesis will be essential in these studies to introduce specific sites for derivatization with structural probes and to slow the decay of intermediates. Thus, combining molecular biology and biophysics will continue to provide solutions to fundamental problems in bR.     

Halide binding by the D212N mutant of Bacteriorhodopsin affects hydrogen bonding of water in the active site

Bacteriorhodopsin (BR), a membrane protein found in Halobacterium salinarum, functions as a light-driven proton pump. The Schiff base region has a quadrupolar structure with positive charges located at the protonated Schiff base and Arg82, and the counterbalancing negative charges located at Asp85 and Asp212. The quadropole inside the protein is stabilized by three water molecules, forming a roughly planar pentagonal cluster composed of these waters and two oxygens of Asp85 and Asp212 (one from each carboxylate side chain). It is known that BR lacks proton-pumping activity if Asp85 or Asp212 is neutralized by mutation, but binding of Cl- has different functional effects in mutants at these positions. Binding of Cl- to D85T converts into a chloride ion pump (Sasaki, J., Brown, L. S., Chon, Y.-S., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J. K. (1995) Science 269, 73-75). On the other hand, photovoltage measurements suggested that binding of Cl- to D212N restores the proton-pumping activity at low pH (Moltke, S., Krebs, M. P., Mollaaghababa, R., Khorana, H. G., and Heyn, M. P. (1995) Biophys. J. 69, 2074-2083). In this paper, we studied halide-bound D212N mutant BR in detail. Light-induced pH changes in a suspension of proteoliposomes containing D212N(Cl-) at pH 5 clearly showed that Cl- restores the proton-pumping activity. Spectral blue-shift induced by halide binding to D212N indicates that halides affect the counterion of the protonated Schiff base, whereas much smaller halide dependence of the lambdamax than in D85T suggests that the binding site is distant from the chromophore. In fact, the K minus BR difference Fourier-transform infrared (FTIR) spectra of D212N at 77 K exhibit little halide dependence for vibrational bands of retinal and protein. The only halide-dependent bands were the C=N stretch of Arg82 and some water O-D stretches, suggesting that these groups constitute a halide-binding pocket. A strongly hydrogen-bonded water molecule is observed for halide-bound D212N, but not for halide-free D212N, which is consistent with our hypothesis that such a water molecule is a prerequisite for proton-pumping activity of rhodopsins. We concluded that halide binding near Arg82 in D212N restores the water-containing hydrogen-bonding network in the Schiff base region. In particular, the ion pair formed by the Schiff base and Asp85 through a strongly hydrogen-bonded water is essential for the proton-pumping activity of this mutant and may be controlled by the halide binding to the distant site.     

Photoproducts of bacteriorhodopsin mutants: a molecular dynamics study.

Molecular dynamics simulations of wild-type bacteriorhodopsin (bR) and of its D85N, D85T, D212N, and Y57F mutants have been carried out to investigate possible differences in the photoproducts of these proteins. For each mutant, a series of 50 molecular dynamics simulations of the photoisomerization and subsequent relaxation process were completed. The photoproducts can be classified into four distinct classes: 1) 13-cis retinal, with the retinal N-H+ bond oriented toward Asp-96; 2) 13-cis retinal, with the N-H+ oriented toward Asp-85 and hydrogen-bonded to a water molecule; 3) 13,14-di-cis retinal; 4) all-trans retinal. Simulations of wild-type bR and of its Y57F mutant resulted mainly in class 1 and class 2 products; simulations of D85N, D85T, and D212N mutants resulted almost entirely in class 1 products. The results support the suggestion that only class 2 products initiate a functional pump cycle. The formation of class 1 products for the D85N, D85T, and D212N mutants can explain the reversal of proton pumping under illumination by blue and yellow light.     

Low temperature FTIR study of the Schiff base reprotonation during the M-to-bR backphotoreaction: Asp 85 reprotonates two distinct types of Schiff base species at different temperatures

We have applied low temperature difference FTIR spectroscopy to investigate intermediates produced from the M intermediate upon blue light excitation (<480 nm). In agreement with an earlier report by Balashov and Litvin (1981), who studied these intermediates with low temperature visible absorption spectrophotometry, we have observed at least three stages in this backphotoreaction. The initial photoproduct is stable at 100 K, and two products of subsequent thermal reactions are observed upon raising the temperature to 130 and 160 K, respectively.

The alterations in the C=N stretching mode of the Schiff base have been identified by isotopically labeling the retinal chromophore, and changes in C=O stretching modes of amino acid residues with acidic side chains have been investigated. Analysis of the C=N stretching mode shows that the Schiff base remains unprotonated after the photochemical reaction at 100 K. Moreover, there are two types of Schiff bases, presumably associated with different bR species, that become thermally reprotonated at 130 and 160 K, respectively. Bands associated with the C=O stretching modes suggest that Asp 85 rather than Asp 96 reprotonates the Schiff base during the M to bR backphotoreaction. This conclusion is consistent with earlier observations that the polarity of electrical signals during this photochemical back reaction is reversed as compared to the thermal regeneration of bR from M.

     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence that ASP-96 deprotonates during the M----N transition

The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96----Ala and Asp-96----Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO- vibration. The Asp-96----Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L----M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N----O or O----bR transition.     

Relocation of internal bound water in bacteriorhodopsin during the photoreaction of M at low temperatures: an FTIR study

Changes in the FTIR difference spectra upon photoconversion of the M intermediate to its photoproduct(s) M' were studied in wild-type bacteriorhodopsin and several mutants at low temperatures. The studies aimed at examining whether internally bound water molecules interact with the chromophore and the key residues Asp85 and Asp96 in M, and whether these water molecules participate in the reprotonation of the Schiff base. We have found that three water molecules are perturbed by the isomerization of the chromophore in the M --> M' transition at 80 K. The perturbation of one water molecule, detected as a bilobe at 3567(+)/3550(-) cm(-)(1), relaxed in parallel with the relaxation of an Asp85 perturbation upon increasing temperature from 80 to 100 and 133 K (before the reprotonation of the Schiff base). Two water bands of M at 3588 and 3570 cm(-)(1) shift to 3640 cm(-)(1) upon photoconversion at 173 K. These bands were attributed to water molecules which are located in the vicinity of the Schiff base and Asp85 (Wat85). In the M to M' transition at 80 and 100 K, where the Schiff base remained unprotonated, the Wat85 pair stayed in similar states to those in M. The reprotonation of the Schiff base at 133 K occurred without the restoration of the Wat85 band around 3640 cm(-)(1). This band was restored at higher temperatures. Two water molecules in the region surrounded by Thr46, Asp96, and Phe219 (Wat219) were perturbed in the M to M' transition at 80 K and relaxed in parallel with the relaxation of the perturbation of Asp96 upon increasing the temperature. Mutant studies show that upon photoisomerization of the chromophore at 80 K one of the Wat219 water molecules moves closer to Val49 (located near the lysine side chain attached to retinal, and close to the Schiff base). These data along with our previous results indicate that the water molecules in the cytoplasmic domain participate in the connection of Asp96 with the Schiff base and undergo displacement during photoconversions, presumably shuttling between the Schiff base and a site close to Asp96 in the L to M to N transitions.     

Asp85 is the only internal aspartic acid that gets protonated in the M intermediate and the purple-to-blue transition of bacteriorhodopsin. A solid-state 13C CP-MAS NMR investigation.

High-resolution solid-state 13C NMR spectra of the ground state and M intermediate of the bacteriorhodopsin mutant D96N with the isotope label at [4-13C]Asp and [11-13C]Trp were recorded. The NMR spectra show that Asp85 is protonated in the M intermediate. The environment of Asp85 is quite hydrophobic. On the other hand, Asp212 remains deprotonated and a slight shift to lower field indicates a more hydrophilic environment. Asp85 also protonates in the purple-to-blue transition of bacteriorhodopsin in the deionized membrane, where it experiences a similar environment to M. The shift of Trp resonances in M reflect a conformational change of the protein in forming the M intermediate.     

Infrared study of the L, M, and N intermediates of bacteriorhodopsin using the photoreaction of M.

Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L/M mixture is separated; at higher temperatures an M/N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-212 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-212, and an additional positive charge in bR.     

Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution.

BACKGROUND: Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump that converts the energy of light into a proton gradient that drives ATP synthesis. The protein comprises seven transmembrane helices and in vivo is organized into purple patches, in which bR and lipids form a crystalline two-dimensional array. Upon absorption of a photon, retinal, which is covalently bound to Lys216 via a Schiff base, is isomerized to a 13-cis,15-anti configuration. This initiates a sequence of events - the photocycle - during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium and reprotonation from the cytoplasmic side. RESULTS: The structure of bR in the ground state was solved to 1.9 A resolution from non-twinned crystals grown in a lipidic cubic phase. The structure reveals eight well-ordered water molecules in the extracellular half of the putative proton translocation pathway. The water molecules form a continuous hydrogen-bond network from the Schiff-base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82. This network is involved both in proton translocation occurring during the photocycle, as well as in stabilizing the structure of the ground state. Nine lipid phytanyl moieties could be modeled into the electron-density maps. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of single crystals demonstrated the presence of four different charged lipid species. CONCLUSIONS: The structure of protein, lipid and water molecules in the crystals represents the functional entity of bR in the purple membrane of the bacteria at atomic resolution. Proton translocation from the Schiff base to the extracellular medium is mediated by a hydrogen-bond network that involves charged residues and water molecules.     

Calculation of proton transfers in Bacteriorhodopsin bR and M intermediates

Residue ionization states were calculated in nine crystal structures of bacteriorhodopsin trapped in bR, early M, and late M states by multiconformation continuum electrostatics. This combines continuum electrostatics and molecular mechanics, deriving equilibrium distributions of ionization states and polar residue and water positions. The three central cluster groups [retinal Schiff base (SB), Asp 85 and Asp 212] are ionized in bR structures while a proton has transferred from SB(+) to Asp 85(-) in late M structures matching experimental results. The proton shift in M is due to weaker SB(+)-ionized acid and more favorable SB(0)-ionized acid interactions following retinal isomerization. The proton release cluster (Glu 194 and Glu 204) binds one proton in bR, which is lost to water by pH 8 in late M. In bR the half-ionized state is stabilized by charge-dipole interactions while full ionization is disallowed by charge-charge repulsion between the closely spaced acids. In M the acids move apart, permitting full ionization. Arg 82 movement connects the proton shifts in the central and proton release clusters. Changes in total charge of the two clusters are coupled by direct long-range interactions. Separate calculations consider continuum or explicit water in internal cavities. The explicit waters and nearby polar residues can reorient to stabilize different charge distributions. Proton release to the low-pH, extracellular side of the protein occurs in these calculations where residue ionization remains at equilibrium with the medium. Thus, the key changes distinguishing the intermediates are indeed trapped in the structures.