Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane.

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M412) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.     

Mechanism of generation and regulation of photopotential by bacteriorhodopsin in bimolecular lipid membrane. The quenching effect of blue light

Photoelectric properties of bacteriorhodopsin incorporated into a bimolecular lipid membrane were investigated with special regard to the mechanism of photoelectric field generation. It was shown that besides its proton pump and electric generator functions bacteriorhodopsin works as a possible molecular regulator of the light-induced membrane potential. When a bimolecular lipid membrane containing bacteriorhodopsin is continuously illuminated in its main visible absorption band, and afterwards by superimposed blue light matching the absorption band of the long-living photobleached bacteriorhodopsin (M₄₁₂) as well, the latter either enhances or decreases the steady-state photoresponse, depending upon the intensity of the green light. Thus, the additional blue-light illumination tends to cause the resultant photoelectric membrane potential to become stabilized. Two alternative schemes are tentatively proposed for the photochemical cycle of bacteriorhodopsin whereby blue light can control photovoltage generation. A kinetic model of the proton pump and the regulation of the photoelectric membrane potential is presented. This model fits all the experimental findings, even quantitatively. From the model some kinetic and physical parameters of this light-driven pump could be determined.     

The electrical response to light of bacteriorhodopsin in planar membranes.

We have measured the light-induced short-circuit current generated by a planar membrane containing bacteriorhodopsin incorporated by vesicle fusion. The experimental results are consistent with an equivalent electrical circuit analogue that assumes that the vesicles remain intact after fusion and that the current generator equivalent of the light-driven proton pump is linearly dependent on bias voltage. The transient response to light of the planar membrane has also been examined. Slow response times are seen to be associated with the capacitive charging and discharging of the fused vesicles. A study of the leading edge of the light response curve of the planar membrane yields information about the transient response of the light-driven proton pump. We propose that the translocation of protons across the membrane is associated with a first-order process characterized by a rate constant lambda.     

Events in proton pumping by bacteriorhodopsin.

The short-circuit photoresponse of a bacteriorhodopsin-based photoactive membrane is studied. The membrane is formed by first coating a Teflon membrane with lipid and then fusing bacteriorhodopsin vesicles to it. An incandescent light source was used to obtain the rise time of the photocurrent in response to a step-function illumination. A fast response, less than 1 ms, characterizes the initial rise and decay of the photocurrent. The trailing edge of the rise and trailing edge of the decay each exhibit different time constants both greater than 1 ms. These slower components show a sensitivity to membrane charging, the presence of diethylether in the bathing solution, and the presence of a charged cation complex in the lipid region. The photoresponse is not analyzed by means of the usual equivalent electrical circuit, but rather in terms of image charges in the conducting electrolyte bathing the membrane. Further experiments using a pulsed laser (pulse width less than 1 microseconds) resolve at least three time constants in the photoresponse: 0.057 ms, 1.06 ms, and 13 ms. Three distinct charge displacements (4.4, 7.5, and 33.1 A) are derived from the data, each corresponding to one of the above time constants.     

Reconstitution of Biological Molecular generators of electric current. Bacteriorhodopsin.

1. Photoinduced generation of electric current by bacteriorhodopsin, incorporated into the planar phospholipid membrane, has been directly measured with conventional electrometer techniques. 2. Two methods for bacteriorhodopsin incorporation have been developed: (a) formation of planar membrane from a mixture of decane solution of phospholipids and of the fraction of violet fragments of the Halobacterium halobium membrane (bacteriorhodopsin sheets), and (b) adhesion of bacteriorhodopsin-containing reconstituted spherical membranes (proteoliposomes) to the planar membrane in the presence of Ca2+ or some other cations. In both cases, illumination was found to induce electric current generation directed across the planar membrane, an effect which was measured by macroelectrodes immersed into electrolyte solutions on both sides of the membrane. 3. The maximal values of the transmembrane electric potential were of about 150 mV at a current of about 10(-11) A. The electromotive force measured by means of counterbalancing the photoeffect by an external battery, was found to reach the value of 300 mV. 4. The action spectrum of the photoeffect coincides with the bacteriorhodopsin absorption spectrum (maximum about 570 nm). 5. Both components of the electrochemical potential of H+ ions (electric potential and delta pH) across the planar membrane affect the bacteriorhodopsin photoelectric response in a fashion which could be expected if bacteriorhodopsin were a light-dependent electrogenic proton pump. 6. La3+ ions were shown to inhibit operation of those bacteriorhodopsin which pump out H+ ions from the La3+-containing compartment. 7. The photoeffect, mediated by proteoliposomes associated with thick planar membrane, is decreased by gramicidin A at concentrations which do not influence the planar membrane resistance in the light. On the contrary, a protonophorous uncoupler, trichlorocarbonylcyanidephenylhydrazone, decreases the photoeffect only if it is added at a concentration lowering the light resistance. The dark resistance is shown to be higher than the light one, and decreases to the light level by gramicidin. 8. A simple equivalent electric scheme consistent with the above results has been proposed.     

A gentle method for the lysis of oral streptococci.

Black lipid planar membranes were prepared by incorporating polymers such as polystyrene in a membrane forming solution. The polymerincorporated planar membranes showed greater stability to applied electric fields and have longer lifertimes. Photopotentials were studied under several conditions; with bacteriorhodopsin in the planar membrane; with bacteriorhodopsin in liposomes; with bacteriorhodopsin fragments in suspension; and with bacteriorhodopsin both in the planar membrane and in liposomes. Skulachev's laboratory has reported that bacteriorhodopsin-liposomes develop potentials across a black lipid planar membrane. In our studies, because the polymer incorporated planar membranes are very stable, it was possible to obtain somewhat larger photopotentials in the presence of bacteriorhodopsin ranging between 30–500 mV. The enhancement of bacteriorhodopsin catalyzed photopotentials, found in the presence of Ca⁺⁺ (or other bivalent cations) and/or applied electric fields, appeared to result from an orientation effect of bacteriorhodopsin at the membrane interface.     

A measurement of the proton pump current generated by bacteriorhodopsin in black lipid membranes

The light-induced electrical current generated by black lipid membranes containing bacteriorhodopsin from Halobacterium halobium has been measured directly. It is shown that a measurement of membrane potential can also be used to obtain the proton pump current developed during illumination. Evidence is presented that the charge movement across the membrane is associated with the release of protons in the photoreaction cycle of bacteriorhodopsin. The time variation of the pump current when the light is turned on suggests the rapid depopulation of some initially occupied state.     

Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes.

The transport activity of purified Na+K+-ATPase was investigated by measuring the electrical pump current induced on black lipid membranes. Discs containing purified Na+K+-ATPase from pig kidney were attached to planar lipid bilayers in a sandwich-like structure. After the addition of only microM concentrations of an inactive photolabile ATP derivative [P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate, caged ATP] ATP was released after illumination with u.v.-light, which led to a transient current in the system. The transient photoresponse indicates that the discs and the underlying membrane are capacitatively coupled. Stationary pump currents were obtained after the addition of the H+, Na+ exchanging agent monensin together with valinomycin to the membrane system, which increased the permeability of the black lipid membrane for the pumped ions. In the absence of ADP and Pi the half saturation for the maximal photoeffect was obtained at 6.5 microM released ATP. The addition of ADP decreased the pump activity. Pump activity was obtained only in the presence of Mg2+ together with Na+ and Na+ and K+. No pump current was obtained in the presence of Mg2+ together with K+. The electrical response was blocked completely by the Na+K+-ATPase-specific inhibitors vanadate and ouabain. No pump currents were observed with a chemically modified protein, which was labelled on the ATP binding site with fluoresceine isothiocyanate. The method described offers the possibility of investigating by direct electrical measurements ion transport of Na+K+-ATPase with a large variety of different parameters.     

Proteoliposomes with right-side-out oriented purple membrane/bacteriorhodopsin require cations inside for proton pumping

Proteoliposomes were prepared by sonication of phospholipids and blue membranes (cation-free purple membranes carrying little activity of light-driven proton pumping) in an acidic medium of very low ionic strength. The majority of the bacteriorhodopsin population in these proteoliposomes was in the right-side-out (as in living cells) orientation as judged from the resultant polypeptides after papain digestion. By raising the pH of sonication, the population of right-side-out oriented bacteriorhodopsin decreased, and consequently that of the inversely oriented one increased. In KCl and NaCl up to certain concentrations or in choline chloride even at high concentrations, in the light, the proteoliposomes with right-side-out bacteriorhodopsin did not pump protons, whereas those with inversely oriented bacteriorhodopsin did. The former began to pump only after cations were likely incorporated/permeated into the proteoliposome and reached the carboxyl terminal (cytosol) side of bacteriorhodopsin/purple membrane.     

The essential role of specific Halobacterium halobium polar lipids in 2D-array formation of bacteriorhodopsin.

The mechanism whereby bacteriorhodopsin (BR), the light driven proton pump from the purple membrane of Halobacterium halobium, arranges in a 2D-hexagonal array, has been studied in bilayers containing the protein, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and various fractions of H. halobium membrane lipids, by freeze fracture electron microscopy and examination of optical diffractograms of the micrographs obtained. Electron micrographs of BR/DMPC complexes containing the entire polar lipid component of H. halobium cell membranes or the total lipid component of the purple membrane, with a protein-to-total lipid molar ratio of less than 1:50 and to which 4 M NaCl had been added, revealed that trimers of BR formed into an hexagonal 2D-array similar to that found in the native purple membrane, suggesting that one or more types of the purple membrane polar lipids are required for array formation. To support this suggestion, bacteriorhodopsin was purified free of endogenous purple membrane lipids and reconstituted into lipid bilayer complexes by detergent dialysis. The lipids used to form these complexes are 1,2-dimyristoyl-sn-glycerol-phosphocholine (DMPC) as the major lipid and, separately, each of the individual lipid types from the H. halobium cell membranes, namely 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-phosphate (DPhPGP), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol 1'-sulphate (DPhPGS), 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol (DPhPG) and 2,3-di-O-phytanyl-1-O-[beta-D-Galp-3-sulphate-(1----6)-alpha-D- Manp-(1----2)-alpha-D-Glcp]-sn-glycerol (DPhGLS). When examined by freeze-fracture electron microscopy, only the complexes containing 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol- 1'-phosphate or 2,3-di-O-phytanyl-sn-glycero-1-phosphoryl-3'-sn-glycerol-1'-sulphate, at high protein density (less than 1:50, bacteriorhodopsin/phospholipid, molar ratio) and to which 4 M NaCl had been added, showed well defined 2D hexagonal arrays of bacteriorhodopsin trimers similar to those observed in the purple membrane of H. halobium.     

Nonlinear Voltage Dependence of the Light-Driven Proton Pump Current of Bacteriorhodopsin

The light-driven proton pump current generated by bacteriorhodopsin reconstituted in asymmetric planar bilayer membranes was investigated. The current-voltage dependence was found to be nonlinear and can be approximated by an exponential at least below +50 mV. The current changed e-fold when the membrane potential was changed by 80 mV. The voltage dependence was analyzed in terms of a barrier model. This analysis revealed an effective displacement of 0.63 elementary charges across the membrane during the rate-limiting step. Comparison of this value with the results from flash-induced photovoltage signals suggests that one proton is pumped per cycle.     

Functional cell-free synthesis of a seven helix membrane protein: in situ insertion of bacteriorhodopsin into liposomes

The expression of membrane proteins for functional and structural studies or medicinal applications is still not very well established. Membrane-spanning proteins that mediate the information flow of the extracellular side with the interior of the cell are prime targets for drug development methods that would allow screening techniques or high throughput formats are of particular interest. Here we describe a systematic approach to the liposome-assisted cell-free synthesis of functional membrane proteins. We demonstrate the synthesis of bacteriorhodopsin (bR(cf)) in presence of small unilamellar liposomes. The yield of bR(cf) per volume cell culture is comparable to that of bacteriorhodopsin in its native host. The functional analysis of bR(cf) was performed directly using the cell-free reaction mixture. Photocycle measurements reveal kinetic data similar to that determined for bR in Halobacterium salinarum cell-envelope vesicles. The liposomes can be attached directly to black lipid membranes (BLM), which allows measuring light activated photocurrents in situ. The results reveal a functional proton pump with properties identical to those established for the native protein.     

Moist and soft, dry and stiff: a review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum

Twenty-five years of neutron experiments on hydration and thermal dynamics in purple membranes of Halobacterium salinarum are reviewed. Neutron diffraction, elastic and quasielastic scattering, allowed to map the distribution of water and lipids and to measure thermal fluctuations and correlation times in the membranes, under various conditions of temperature, hydration and lipid environment. Strong correlations were established between dynamics parameters and the activity of bacteriorhodopsin (the purple membrane protein), as a light driven proton pump supporting the hypothesis that the influence of hydration on activity is in fact due to its effects on membrane thermal dynamics. Hydrogen-deuterium labelling experiments highlighted stiffer and softer parts in the bacteriorhodopsin structure. The soft parts would allow the conformational changes involved in activity, while the stiffer ones may control a valve-like function in vectorial proton transfer.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

pH对菌紫质分子的旋转运动和光电响应的影响

用闪光诱导瞬间二向色性方法测量了不同pH条件下的菌紫质分子在脂质囊泡中的旋转扩散运动.在人工平扳膜(BLM)系统中测量了不同pH条件下菌紫质分子的光电响应.在pH3至8.3的范围内没有明显观察到菌紫质分子在膜中旋转运动上的差别.pH低于3时,菌紫质分子旋转运动受到影响;pH高于11时,观察不到旋转扩散运动.在BLM系统中测量了pH2到pH11范围内菌紫质分子的光电响应信号,随着pH的增加,无论紫膜碎片还是单体菌紫质分子的光电响应逐渐由照光后快速正信号并快速衰减及撤光时的快速负信号并逐渐衰减变成慢的正信号.pH高于9.4时,单体菌紫质分子的光电响应信号由正变负,pH高于11时,观察不到信号.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

Light-induced generation of a protonmotive force and Ca2+-transport in membrane vesicles of Streptococcus cremoris fused with bacteriorhodopsin proteoliposomes

The light-driven primary proton pump bacteriorhodopsin has been incorporated in the cytoplasmic membrane of Streptococcus cremoris, in order to generate a protonmotive force across these membranes. This has been achieved by fusion of S. cremoris membrane vesicles with bacteriorhodopsin proteoliposomes. This fusion occurred when both preparations were mixed at low pH (less than 6.0), as shown by sucrose density gradient centrifugation and by dilution of fluorescent phospholipids incorporated into the bacteriorhodopsin proteoliposomes. Fusion was strongly enhanced by the presence of negatively charged phospholipids in the liposomal bilayer. When proteoliposomes were used that showed light-dependent proton uptake, the orientation of bacteriorhodopsin in the fused membranes was inside-out with respect to the in vivo orientation in Halobacterium halobium. Consequently, in the light a ΔΨ, interior positive and a ΔpH, interior acid were generated. This protonmotive force could drive calcium uptake in the fused membranes. The uptake increased hyperbolically with increasing light intensity and was abolished by bleaching of bacteriorhodopsin. Addition of the ionophore valinomycin stimulated calcium uptake and led to an increase of the ΔpH. Calcium uptake was strongly decreased in the dark and in the light in the presence of uncouplers, nigericin or both valinomycin and nigericin.     

Production of functional bacteriorhodopsin by an Escherichia coli cell‐free protein synthesis system supplemented with steroid detergent and lipid

Cell‐free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis‐based Escherichia coli cell‐free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light‐driven proton pump bacteriorhodopsin, consisting of seven transmembrane α‐helices. The cell‐free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3–0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent‐lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.