Proton Pump Bacteriorhodopsin Research Articles

Azide Reduces the Hydrophobic Barrier of the Bacteriorhodopsin Proton Channel

The sensitivity of a nitroxide spin label to the polarity of its environment has been used to estimate the hydrophobic barrier of the proton channel of the transmembrane proton pump bacteriorhodopsin. By means of site-specific mutagenesis, single cysteine residues were introduced at 10 positions located at the protein surface, in the protein interior, and along the proton pathway. After reaction with a methanethiosulfonate spin label, the principle values of the hyperfine tensor A and the g-tensor were determined from electron paramagnetic resonance spectra measured at 170 K. The shape of the hydrophobic barrier of the proton channel is characterized in terms of a polarity index, Δ A, determined from the variation of the hyperfine coupling constant A zz. The maximum of the hydrophobic barrier is found to be close to the retinal chromophore in the proton uptake pathway. The effect of the asymmetric distribution of charged and polar residues in the proton release and uptake pathways is clearly reflected in the behavior of the hydrophobic barrier. The presence of azide reduces the barrier height of both the cytoplasmic and extracellular channels. This finding supports the view of azide and other weakly acidic anions as catalysts for the formation of hydrogen-bonded networks in proton pathways of proteins.

Interpretation of the spatial charge displacements in bacteriorhodopsin in terms of structural changes during the photocycle.

We have recently introduced a method, made possible by an improved orienting technique using a combination of electric and magnetic fields, that allows the three-dimensional detection of the intramolecular charge displacements during the photocycle of bacteriorhodopsin. This method generates electric asymmetry, a prerequisite for the detection of electric signal on the macroscopic sample, in all three spatial dimensions. Purple membrane fragments containing bacteriorhodopsin were oriented so that their permanent electric dipole moment vectors were perpendicular to the membrane plane and pointed in the same direction. The resulting cylindrical symmetry was broken by photoselection, i. e., by flash excitation with low intensity linearly polarized light. From the measured electric signals, the three-dimensional motion of the electric charge center in the bacteriorhodopsin molecules was calculated for the first 400 microseconds. Simultaneous absorption kinetic recording provided the time-dependent concentrations of the intermediates. Combining the two sets of data, we determined the discrete dipole moments of intermediates up to M. When compared with the results of current molecular dynamics calculations, the data provided a decisive experimental test for selecting the optimal theoretical model for the proton transport and should eventually lead to a full description of the mechanism of the bacteriorhodopsin proton pump.

1PB014 時間分解蛍光法によるバクテリオロドプシンの光プロトンポンプ作用のin vivo測定

1PB014 時間分解蛍光法によるバクテリオロドプシンの光プロトンポンプ作用のin vivo測定

Understanding Structure and Function in the Light-Driven Proton Pump Bacteriorhodopsin

The atomic structure of bacteriorhodopsin and the outlines of its proton transport mechanism are now available. Photoisomerization of the retinal in the chromophore creates a steric and electrostatic conflict at the retinal binding site. The free energy gain sets off a sequence of reactions in which directed proton transfers take place between the protonated retinal Schiff base, Asp-85, and Asp-96. These internal steps, and other proton transfers at and near the two aqueous interfaces, add up to the translocation of a proton from the cytoplasmic to the extracellular side of the membrane. Bound water plays a crucial role in proton conduction in both extracellular and cytoplasmic regions, but the means by which the protons move from site to site differ. Proton release to the extracellular surface is through interaction of a hydrogen-bonded chain of identified aspartic acid, arginine, water, and glutamic acid residues with Asp-85, while proton uptake from the cytoplasmic surface utilizes a single aspartic acid, Asp-96, whose protonation state appears to be regulated by the protein conformation dependent hydration of this region. The directionality of the translocation is ensured by the accessibility of the Schiff base to the extracellular and cytoplasmic directions after the retinal is photoisomerized, as well as the changing proton affinities of the acceptor Asp-85 and donor Asp-96.

The uncoupling of bacteriorhodopsin by high temperature and anaesthetics

1. General anaesthetics induce reversible structural and functional modifications of the proton pump bacteriorhodopsin contained in the purple membrane of halobacteria. 2. This short paper describes the phenomenological similarity between the effects of anaesthetics and heating on this system. 3. pH dependence and thermal analysis suggest that the structural change taking place at room temperature in the presence of anaesthetic is the same as that observed at temperatures higher than 70°C in the native membrane. In addition, part of the structural modification has an irreversible character when induced by anaesthetic as well as by heating.

Photovoltage evidence that Glu-204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85. pH dependence of the electrogenicity of M intermediate formation in the wild-type bacteriorhodopsin reveals only one component corresponding to the protonation of D85 in the bacteriorhodopsin ground state and transition of the purple neutral form into the blue acid form. Thus, the p K of E204 in the M state is close to the p K of D85 in the bacteriorhodopsin ground state (<3) and far below the p K of the terminal proton release group (∼6). It is concluded that E204 functions as the intermediate proton donor rather than the terminal proton release group in the bacteriorhodopsin proton pump.

Effective Light-Induced Hydroxylamine Reactions Occur with C 13 = C 14 Nonisomerizable Bacteriorhodopsin Pigments

The light-driven proton pump bacteriorhodopsin (bR) undergoes a bleaching reaction with hydroxylamine in the dark, which is markedly catalyzed by light. The reaction involves cleavage of the (protonated) Schiff base bond, which links the retinyl chromophore to the protein. The catalytic light effect is currently attributed to the conformational changes associated with the photocycle of all- trans bR, which is responsible for its proton pump mechanism and is initiated by the all- trans → 13- cis isomerization. This hypothesis is now being tested in a series of experiments, at various temperatures, using three artificial bR molecules in which the essential C 13 C 14 bond is locked by a rigid ring structure into an all- trans or 13- cis configuration. In all three cases we observe an enhancement of the reaction by light despite the fact that, because of locking of the C 13 C 14 bond, these molecules do not exhibit a photocycle, or any proton-pump activity. An analysis of the rate parameters excludes the possibility that the light-catalyzed reaction takes place during the ∼20-ps excited state lifetimes of the locked pigments. It is concluded that the reaction is associated with a relatively long-lived ( μs-ms) light-induced conformational change that is not reflected by changes in the optical spectrum of the retinyl chromophore. It is plausible that analogous changes (coupled to those of the photocycle) are also operative in the cases of native bR and visual pigments. These conclusions are discussed in view of the light-induced conformational changes recently detected in native and artificial bR with an atomic force sensor.

Actinic light and pH effect on the proton pumping of bacteriorhodopsin

The actinic light effect on the bacteriorhodopsin (BR) photocycle kinetics led to the assumption of a cooperative interaction between the photocycling BR molecules. In this paper we report the results of the actinic light effect and pH on the proton release and uptake kinetics. An electrical method is applied to detect proton release and uptake during the photocycle [E. Papp, G. Fricsovszky, J. Photochem. Photobiol. B: Biol. 5 (1990) 321]. The BR photocycle kinetics was also studied by absorption kinetics measurements at 410 nm and the data were analyzed by the local analysis of the M state kinetics [E. Papp, V.H. Ha, Biophys. Chem. 57 (1996) 155]. While at high pH and ionic strength, we found a similar behavior as reported earlier, at low ionic strength the light effect proved to be more complex. The main conclusions are the following: Though the number of BR excited to the photocycle (fraction cycling, fc) goes to saturation with increasing laser pulse energy, the absorbed energy by BR increases linearly with pulse energy. From the local analysis we conclude that the light effect changes the kinetics much earlier, already at the L intermediate state decay. The transient electric signal, caused by proton release and uptake, can be decomposed into two components similarly to the absorption kinetic data of the M intermediate state. The actinic light energy affects mainly the ratio of the two components and the proton movements inside BR while pH has an effect on the kinetics of the proton release and uptake groups at the membrane surface.

Photoactivities of the Red-Shifted Azulenic Bacteriorhodopsin Analogues

Incorporating red-shifted retinal analogues, such as azulenic retinals, into bacterioopsin provides a method to evaluate the low-energy limit for bacteriorhodopsin (bR) proton-pumping activity. We report results of nanosecond time-resolved studies of three azulenic bR analogues. In contrast to a recent report on an azulenic bR analogue (Druzhko et al., 1996),4 we conclude that such pigments do not exhibit photocycles (no detectable intermediates) and suspect that the preferred direction of deactivation is internal conversion to the low-lying forbidden S1 state of the azulene chromophore. We further demonstrated that for slow-binding bR analogues, the samples can easily be contaminated with small amounts of native bR.

Effects of substitutions D73E, D73N, D103N and V106M on signaling and pH titration of sensory rhodopsin II.

Several mutations in the repellent phototaxis receptor sensory rhodopsin II (SRII), in residues homologous to residues important in the related proton pump bacteriorhodopsin, were expressed in Pho81Wr-, a Halobacterium salinarum strain deficient in production of SRII and its transducer protein HtrII. The lack of production of SRII and HtrII is shown to be due to insertion of an ISH2 transposon into the promoter region upstream of the htrII-sopII gene pair. Near wild-type phototaxis responses are rescued in Pho81Wr- by expression of HtrII with D73E, D103N or V106M receptors. Partial responses are restored by the HtrII-D73N pair. From absorption spectroscopy of his-tag-purified receptor protein from mutants D73N and D73E we conclude that Asp73 is the primary counterion to the protonated Schiff base in SRII, like the corresponding Asp85 in bacteriorhodopsin. The absorption maximum of SRII (487 nm) is shifted to 514 nm in mutant D73N, a 1080 cm-1 shift identical to that caused by D85N in bacteriorhodopsin. Acid titration of SRII also induces the red shift with a pK of 3.0 in wild type. The absorption shift and the pK are nearly the same in V106M and D103N, but the pK is raised to 5.1 in D73E, confirming that Asp73 is the residue responsible for this spectral transition.

Titration kinetics of Asp-85 in bacteriorhodopsin: exclusion of the retinal pocket as the color-controlling cation binding site

The spectrum (the purple↔blue transition) and function of the light-driven proton pump bacteriorhodopsin are determined by the state of protonation of the Asp-85 residue located in the vicinity of the retinal chromophore. The titration of Asp-85 is controlled by the binding/unbinding of one or two divalent metal cations (Ca 2+ or Mg 2+). The location of such metal binding site(s) is approached by studying the kinetics of the cation-induced titration of Asp-85 using metal ions and large molecular cations, such as quaternary ammonium ions, R 4N + (R=Et, Pr, a divalent `bolaform ion' [Et 3N +-(CH 2) 4-N +Et 3] and the 1:3 molecular complex formed between Fe 2+ and 1,10-phenanthroline (OP). The basic multi-component kinetic features of the titration, extending from 10 −2 to 10 4 s, are unaffected by the charge and size of the cation. This indicates that cation binding to bR triggers the blue→purple titration in a fast step, which is not rate-determining. In view of the size of the cations involved, these observations indicate that the cation binding site is in an exposed location on, or close to, the membrane surface. This excludes previous models, which placed the color-controlling Ca 2+ ion in the retinal binding pocket.

Fourier transform infrared double-flash experiments resolve bacteriorhodopsin's M1 to M2 transition

The orientation of the central proton-binding site, the protonated Schiff base, away from the proton release side to the proton uptake side is crucial for the directionality of the proton pump bacteriorhodopsin. It has been proposed that this movement, called the reprotonation switch, takes place in the M1 to M2 transition. To resolve the molecular events in this M1 to M2 transition, we performed double-flash experiments. In these experiments a first pulse initiates the photocycle and a second pulse selectively drives bR molecules in the M intermediate back into the BR ground state. For short delay times between initiating and resetting pulses, most of the M molecules being reset are in the M1 intermediate, and for longer delay times most of the reset M molecules are in the M2 intermediate. The BR-M1 and BR-M2 difference spectra are monitored with nanosecond step-scan Fourier transform infrared spectroscopy. Because the Schiff base reprotonation rate is kM1 = 0.8 x 10(7) s(-1) in the light-induced M1 back-reaction and kM2 = 0.36 x 10(7) s(-1) in the M2 back-reaction, the two different M intermediates represent two different proton accessibility configurations of the Schiff base. The results show only a minute movement of one or two peptide bonds in the M1 to M2 transition that changes the interaction of the Schiff base with Y185. This backbone movement is distinct from the larger one in the subsequent M to N transition. No evidence of a chromophore isomerization is seen in the M1 to M2 transition. Furthermore, the results show time-resolved reprotonation of the Schiff base from D85 in the M photo-back-reaction, instead of from D96, as in the conventional cycle.

Microsecond atomic force sensing of protein conformational dynamics: implications for the primary light-induced events in bacteriorhodopsin.

In this paper a new atomic force sensing technique is presented for dynamically probing conformational changes in proteins. The method is applied to the light-induced changes in the membrane-bound proton pump bacteriorhodopsin (bR). The microsecond time-resolution of the method, as presently implemented, covers many of the intermediates of the bR photocycle which is well characterized by spectroscopical methods. In addition to the native pigment, we have studied bR proteins substituted with chemically modified retinal chromophores. These synthetic chromophores were designed to restrict their ability to isomerize, while maintaining the basic characteristic of a large light-induced charge redistribution in the vertically excited Franck-Condon state. An analysis of the atomic force sensing signals lead us to conclude that protein conformational changes in bR can be initiated as a result of a light-triggered redistribution of electronic charge in the retinal chromophore, even when isomerization cannot take place. Although the coupling mechanism of such changes to the light-induced proton pump is still not established, our data question the current working hypothesis which attributes all primary events in retinal proteins to an initial trans<==>cis isomerization.

Neutron diffraction on purple membranes essential water molecules in the light-driven proton pump bacteriorhodopsin

Neutron diffraction and time-resolved laser-spectroscopic experiments on the light-driven proton-pump bacteriorhodopsin revealed the location of water molecules and their functional importance. About seven water molecules in the proton pathway across this membrane protein were determined at 15% rel. humidity in the ground state.

Exploring an antifungal target in the plasma membrane H +-ATPase of fungi

The plasma membrane H +-ATPase is a promising new antifungal target that is readily probed with the sulfhydryl-reactive reagent omeprazole. Inhibition of the H +-ATPase by omeprazole is closely linked to cell killing, and it has been suggested that enzyme inhibition may result from a covalent interaction within the first two transmembrane segments (M1 and M2) (Monk et al. (1995) Biochim. Biophys. Acta 1239, 81–90). In this study, the molecular nature of this interaction was examined by screening a series of 26 well-characterized pma1 mutations residing in the first two transmembrane segments of the H +-ATPase from Saccharomyces cerevisiae. Only two pma1 mutants, A135G and G158D,G156C, were found to significantly decrease the sensitivity of cells for omeprazole. In contrast, enhanced sensitivity was observed at a number of positions, with D140C(A) and M128C producing the most significant increases in sensitivity. The introduction of cysteine at various locations within this region only marginally affected omeprazole sensitivity, suggesting that this region was not a direct site of covalent modification. Rather, its conformation influences omeprazole binding at some other locus. In order to determine the sidedness of the omeprazole interaction, a novel in vitro assay system was exploited that utilized liposomes co-reconstituted with the H +-ATPase and the light-driven proton pump bacteriorhodopsin. Omeprazole was found to completely inhibit proton transport by the H +-ATPase at 50 μM in this system. An asymmetrically-distributed chemical trap system involving glutathione was used to demonstrate that this inhibition appears localized to the extracellular portion of the enzyme. This work indicates that omeprazole can inhibit the H +-ATPase from its extracellular face, and this inhibition is influenced by changes in the M1, M2 region of the protein.

Molecular Reaction Mechanisms of Proteins Monitored by Nanosecond Step-Scan FT-IR Difference Spectroscopy

Step-scan FT-IR difference spectroscopy with 100-ns time resolution is applied to the membrane protein bacteriorhodopsin. The experimetal setup and data acquisition are described in great detail. The IR absorbance changes accompanying bacteriorhodopsin's photocycle are presented in a three-dimensional representation. The data quality allows one, for example, to resolve the biphasic rise of the photocycle-intermediate M. A bacteriorhodopsin's proton pump model based on the FT-IR works is presented.

Time-resolved fourier transform infrared study of structural changes in the last steps of the photocycles of Glu-204 and Leu-93 mutants of bacteriorhodopsin.

The last intermediate in the photocycle of the light-driven proton pump bacteriorhodopsin is the red-shifted O state. The structure and dynamics of the last step in the photocycle were characterized with time-resolved Fourier transform infrared spectroscopy of the mutants of Glu-204 and Leu-93, which accumulate this intermediate in much larger amounts than the wild type. The results show that E204Q and E204D give distorted all-trans-retinal chromophore like the O intermediate of the wild type. This is simply due to the perturbation of the proton acceptor function of Glu-204 in the O-to-BR transition in the Glu-204 mutants. The corresponding red-shifted intermediates of L93M, L93T, and L93S have a 13-cis chromophore like the N intermediate of the wild type, as reported from analysis of extracted retinal [Delaney, J. K., Schweiger, U., & Subramaniam, S. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11120-11124]. In spite of their different chromophore structures from the O intermediate, the red-shifted intermediates are similar to the O intermediate but not to the N intermediate of the wild type with respect to structural changes in the peptide carbonyls. The structural changes around Asp-96 in the N intermediate are completely restored also in the red-shifted intermediates of the Leu-93 mutants like in the O intermediate. These results imply that the protein structural changes in the last step proceed regardless of thermal isomerization of the chromophore. Time-resolved Fourier transform infrared spectroscopy with the Glu-204 mutants suggests that the response of Asp-204 (Glu-204 in the wild type) to the protonation of Asp-85 during formation of the M intermediate, which results in proton release, is slow and may occur through structural changes.

Intramembrane signaling mediated by hydrogen-bonding of water and carboxyl groups in bacteriorhodopsin and rhodopsin.

The light-induced mechanism for proton pumping of bacteriorhodopsin was studied by Fourier transform infrared spectroscopy of the discrete sequential intermediate states, L, M, and N. Attention is focused on L in the early microsecond time range, as a transition state in which the Schiff base forms strong H-bonding with a water molecule coordinated with Asp85. This structure leads to transfer of the Schiff base proton to Asp85 in the L-to-M process, which then triggers proton release from Glu204 to the extracellular surface. H-bonding of Arg82 and water molecules are involved in this process. Chloride can replace Asp85 in the D85T mutant, and this anion will be then transported instead of a proton. In L, structural perturbations are induced also around Asp96, through a string of H-bonding mediated by internal water molecules and peptide carbonyls in helices B and C, and Trp182 in helix F. These may cause the structural changes that occur later in the M-to-N process. Similar interactions, through internal water molecules and the peptide bonds in helices B and C, take place in bovine rhodopsin. They transduce changes across the membrane from the Schiff base to the cytoplasmic surface, where the activation of the transducin occurs.

Role of Calcium in the Proton Pump of Bacteriorhodopsin. Microwave Evidence for a Cation-Gated Mechanism

Bacteriorhodopsin is the light-activated proton pump in the purple membrane of Halobacterium salinarium. One aspect of the proton pump mechanism that remains to be fully revealed is how back-translocation of the proton is eliminated despite the fact that gradients of 250 mV or more can exist in favor of reverse proton motion. This mechanistic component is examined with reference to the static and photodynamic resonances observed in the 1−40 GHz region of the microwave spectrum. The light-adapted form of bacteriorhodopsin displays strong microwave absorption with maxima near 8 and 25 GHz. We identify the origin of the microwave activity to protein-bound divalent cations by noting that (1) the ion-free blue membrane is microwave inactive, and (2) the microwave activity of the protein can be regenerated by adding calcium and/or magnesium to the protein. Initiation of the photocycle generates additional microwave activity in the low-frequency region of the spectrum. The M minus bR difference spectrum shows a single-cation resonance at 9.8 GHz. We assign this resonance to the release of a calcium ion from the chromophore-adjacent binding site into the proton channel. We conclude that a key biological role of the chromophore-adjacent calcium binding site is to provide a divalent cation gate that not only facilitates chromophore reprotonation but also prevents back-transfer of the proton during the latter stages of the photocycle.

P-E4-05 Actinic light effect on the proton pump of bacteriorhodopsin: Papp E., Ha V. H., Ablonczy ZS. Dept. of Atomic Physics, Eötvös University, Budapest (H)

P-E4-05 Actinic light effect on the proton pump of bacteriorhodopsin: Papp E., Ha V. H., Ablonczy ZS. Dept. of Atomic Physics, Eötvös University, Budapest (H)