Temperature dependence of photovoltages generated by bacteriorhodopsin.

The temperature dependence of the photovoltage developed by a model membrane containing bacteriorhodopsin (BR) is studied. The model membrane is formed by first coating a thin Teflon sheet with lipid and then fusing BR vesicles to it. The time course of the photoresponse is resolved down to 1 microsecond. The photoresponse is taken to be a sum of exponentials. Exponential time constants and amplitudes are determined by an analysis of the photoresponse with a photovoltage vs. log time plot, correlation filter, and nonlinear least-squares routine. The photovoltage is taken to be the sum of three exponentials but only two of the three time constants are resolved. Both are temperature dependent and indicate a thermally activated transport process. The corresponding activation energies are 55 kJ/mol and 62 kJ/mol. Since the photovoltage is proportional to charge times displacement the corresponding charge displacements are 11 and 34 A assuming a total displacement of 45 A. The remaining exponential term corresponds to a small negative transient in the photovoltage that has a rise time less than 1 microsecond even at -20 degrees C. The calculated charge displacement is estimated to be less than 2 A.     

Photocurrents generated by bacteriorhodopsin on planar bilayer membranes

When purple-membrane fragments from Halobacterium halobium are added to one aqueous phase of a positively-charged black lipid membrane, the membrane becomes photoelectrically active. Under normal conditions the steady-state photo-current is extremely low, but increases considerably when the lipid bilayer is doped with proton-permeable gramicidin channels or with a lipophilic acid-base system. These findings indicate that the purple-membrane sheets are bound to the surface of the bilayer, forming a sandwich-like structure. The time-behaviour of the photocurrent may be interpreted on the basis of a simple equivalent circuit which contains the conductance and capacitance of the purple membrane in series with the conductance and capacitance of the lipid bilayer. From the dependence of the photocurrent on the polarization of the exciting light the average angle between the transition moment of the retinal chromophore and the plane of the bilayer was calculated to be about 28 degrees. Furthermore, it was shown that chromophore-free apomembrane binds to the lipid bilayer and that its photoelectrical activity can be restored in situ by adding all-trans-retinal to the aqueous phase.     

Photocurrents generated by bacteriorhodopsin adsorbed on nano-black lipid membranes

Purple membranes were adsorbed on freestanding lipid bilayers, termed nano-black lipid membranes (nano-BLMs), suspending the pores of porous alumina substrates with average pore diameters of 280 nm. Nano-BLMs were obtained by first coating the upper surface of the highly ordered porous alumina substrates with a thin gold layer followed by chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine and octadecylamine dissolved in n-decane onto the hydrophobic submonolayer. By means of impedance spectroscopy, the quality of the nano-BLMs was verified. The electrical parameters confirm the formation of single lipid bilayers with high membrane resistances covering the porous matrix. Adsorption of purple membranes on the nano-BLMs was followed by recording the photocurrents generated by bacteriorhodopsin upon continuous light illumination. The membrane system exhibits a very high long-term stability with the advantage that not only transient but also stationary currents are recordable. By adding the proton ionophore carbonyl cyanide-m-chlorophenylhydrazone the conductivity of the nano-BLMs increases, resulting in a higher stationary current, which proves that proton conductance occurs across the nano-BLMs.     

Ion transport through liquid membrane by cyclic octapeptides

Cation transport through a chloroform liquid membrane by cyclic octapeptides—cyclo(Leu-Pro)₄, cyclo(Phe-Pro)₄, and cyclo[Lys(Z)-Pro]₄—was investigated. All of these cyclic octapeptides transported K⁺ and Ba²⁺, and the rate of cation transport was correlated with the ability to extract cations from the aqueous phase to the chloroform phase. Among them, cyclo (Leu-Pro)₄ was the most efficient and transported K⁺ and Ba²⁺ selectively from other alkali and alkaline earth cations, respectively. The rate of K⁺ transport by cyclo(Leu-Pro)₄ was about one-third as fast as that by dicyclohexyl 18-crown-6. Picrate anion transport against its concentration gradient was observed by cyclo(Leu-Pro)₄, which is conjugated with the selective transport of K⁺. Complex formation in a liposome between cyclo(Leu-Pro)₄ and Ba²⁺ was observed, but the binding constant was low.     

Insertion of bacteriorhodopsin into polymerized diacetylenic phosphatidylcholine bilayers

We have developed a method to incorporate the membrane protein bacteriorhodopsin into polymerized bilayers composed of a diacetylenic phosphatidylcholine, 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and a non-polymerizable phospholipid, dinonanoylphosphatidylcholine (DNPC). The extent of DC8,9PC polymerization in the bilayer was significantly improved when 2:1 mole ratio DNPC-DC8,9PC was used. Octyl glucopyranoside-solubilized bacteriorhodopsin was inserted into the polymerized DNPC-DC8,9PC bilayers by overnight incubation at 4 degrees C followed by dialysis to remove the detergent. The protein was inserted into the membranes after photo-polymerization to avoid inactivation of the protein due to the UV irradiation. The insertion of bacteriorhodopsin into the polymerized DNPC-DC8,9PC membranes was confirmed by density gradient centrifugation, UV/visible spectroscopy, and freeze fracture electron microscopy. The polymerized DNPC-DC8,9PC membranes containing bacteriorhodopsin were about 10% protein by weight. These results suggest that mixed lipid systems such as the DNPC-DC8,9PC can be used to improve both the extent of polymerization and the efficiency of membrane protein incorporation in the polymerized bilayer.     

Influence of the intrinsic membrane protein bacteriorhodopsin on gel-phase domain topology in two-component phase-separated bilayers.

We have investigated the effect of the intrinsic membrane protein bacteriorhodopsin of Halobacterium halobium on the lateral organization of the lipid phase structure in the coexistence region of an equimolar mixture of dimyristoylphos-phatidylcholine and distearoylphosphatidylcholine. The fluorescence recovery after photobleaching (FRAP) technique was used to monitor the diffusion of both a lipid analog (N-(7-nitrobenzoxa-2,3-diazol-4-yl)-dimyristoylphosphatidyle thanolamine, NBD-DMPE) and fluorescein-labeled bacteriorhodopsin (Fl-BR). In the presence of bacteriorhodopsin, the mobile fractions of the two fluorescent probes display a shift of the percolation threshold toward lower temperatures (larger gel-phase fractions), independent of the protein concentration, from 43 degrees C (without bacteriorhodopsin) to 39 degrees C and 41 degrees C for NBD-DMPE and Fl-BR, respectively. Moreover, in the presence of bacteriorhodopsin, the gel-phase domains are much less efficient in restricting the diffusion of both probes than they are in the absence of the protein in the two-phase coexistence region. Bacteriorhodopsin itself, however, obstructs diffusion of NBD-DMPE and Fl-BR to about the same extent in the fluid phase of the two-phase region as it does in the homogeneous fluid phase. These observations suggest that 1) the protein induces the formation of much larger and/or more centrosymmetrical gel-phase domains than those formed in its absence, and 2) bacteriorhodopsin partitions almost equally between the coexisting fluid and gel phases. Although the molecular mechanisms involved are not clear, this phenomenon is fully consistent with the effect of the transmembrane peptide pOmpA of Escherichia coli investigated by electron spin resonance in the same lipid system.     

Molecular sorting of lipids by bacteriorhodopsin in dilauroylphosphatidylcholine/distearoylphosphatidylcholine lipid bilayers.

A combined experimental and theoretical study is performed on binary dilauroylphosphatidylcholine/distearoylphosphatidylcholine (DLPC/DSPC) lipid bilayer membranes incorporating bacteriorhodopsin (BR). The system is designed to investigate the possibility that BR, via a hydrophobic matching principle related to the difference in lipid bilayer hydrophobic thickness and protein hydrophobic length, can perform molecular sorting of the lipids at the lipid-protein interface, leading to lipid specificity/selectivity that is controlled solely by physical factors. The study takes advantage of the strongly nonideal mixing behavior of the DLPC/DSPC mixture and the fact that the average lipid acyl-chain length is strongly dependent on temperature, particularly in the main phase transition region. The experiments are based on fluorescence energy transfer techniques using specifically designed lipid analogs that can probe the lipid-protein interface. The theoretical calculations exploit a microscopic molecular interaction model that embodies the hydrophobic matching as a key parameter. At low temperatures, in the gel-gel coexistence region, experimental and theoretical data consistently indicate that BR is associated with the short-chain lipid DLPC. At moderate temperatures, in the fluid-gel coexistence region, BR remains in the fluid phase, which is mainly composed of short-chain lipid DLPC, but is enriched at the interface between the fluid and gel domains. At high temperatures, in the fluid phase, BR stays in the mixed lipid phase, and the theoretical data suggest a preference of the protein for the long-chain DSPC molecules at the expense of the short-chain DLPC molecules. The combined results of the experiments and the calculations provide evidence that a molecular sorting principle is active because of hydrophobic matching and that BR exhibits physical lipid selectivity. The results are discussed in the general context of membrane organization and compartmentalization and in terms of nanometer-scale lipid-domain formation.     

Proton transport by bacteriorhodopsin through an interface film

Summary Interface films of purple membrane and lipid containing spectroscopically intact and oriented bacteriorhodopsin have been used as a model system to study the function of this protein. Small positive charges in surface potential (<1 mV) are detected upon illumination of these films at the air-water interface. These photopotentials, are not affected by overlaying the interface film with a thin layer (0.3 mm) of decane. However, they are dramatically increased when lipid soluble proton carriers FCCP or DNP are added to the decane. The polarity of the photopotential indicates that, in the light, positive charges are transported through the interface from the aqueous to the organic phase. The action spectrum of the photopotential is identical to the absorption spectrum of bacteriorhodopsin. Since bacteriorhodopsin molecules are oriented with their intracellular surface towards the aqueous subphase, the characteristics of the photopotential indicate that in the light bacteriorhodopsin translocates protons from its intracellular to its extracellular surface. The kinetics of the photopotential reveal that the rate and extent of proton transport are proportional both to the fraction of bacteriorhodopsin molecules excited and to the concentration of proton acceptor. The photopotentials result from changes in the ionic distribution across the decane-water interface and can be cancelled by lipid soluble anions.     

Pair distribution functions of bacteriorhodopsin and rhodopsin in model bilayers.

The pair distribution functions have been measured from freeze-fracture pictures of bacteriorhodopsin and rhodopsin recombinants with diacyl phosphatidylcholines (PC) of various hydrocarbon chain lengths. Pictures were used of samples that had been frozen from above the phase transition temperature of the lipid. Measured functions were compared with those calculated from two model interparticle potential energy functions, (a) a hard-disk repulsion only, and (b) a hard-disk repulsion plus electrostatic repulsion for a point charge buried in the membrane. The measured functions for bacteriorhodopsin di 12:0 PC, di 14:0 PC, and di 16:0 PC recombinants can be simulated using an interparticle hard-disk repulsion only. Bleached rhodopsin di 12:0 PC and di 18:1 trans-PC recombinants, and dark-adapted rhodopsin di 10:0 PC recombinants yield functions that are better simulated by assuming an additional repulsive interaction. The measured functions resemble those calculated using the hard-disk plus electrostatic repulsion model. The picture of dark-adapted rhodopsin in di 18:1 trans-PC frozen from 20 degrees C shows partial aggregation that is apparent in the measured pair distribution function. This attractive interaction persists even at 37 degrees C, where the measured function shows deviations from the hard-disk repulsive model, indicative of an attractive interparticle interaction. Implications of these results are discussed in terms of protein-lipid interactions.     

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.     

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.     

Influence of membrane structure on the kinetics of carrier-mediated ion transport through lipid bilayers

Charge-pulse relaxation experiments of valinomycin-mediated Rb⁺ transport have been carried out in order to study the influence of membrane structure on carrier kinetics. From the experimental data the rate constants of association (k_R) and dissociation (k_D) of the ion-carrier complex as well as the rate constants of translocation of the complex (k_MS) and of the free carrier (k_S) could be obtained. The composition of the planar bilayer membrane was varied in a wide range. In a first series of experiments, membranes made from glycerolmonooleate dissolved in different n-alkanes (n-decane to n-hexadecane), as well as solvent-free membranes made from the same lipid by the Montal-Mueller technique were studied. The translocation rate constants k_S and k_MS were found to differ by less than a factor of two in the membranes of different solvent content. Much larger changes of the rate constants were observed if the structure of the fatty acid residue was varied. For instance, an increase in the number of double bonds in the C₂₀ fatty acid from one to four resulted in an increase of k_S by a factor of seven and in an increase of k_MS by a factor of twenty-four. The stability constant K = k_R/k_D of the ion-carrier complex as well as the translocation rate constants k_S and k_MS were found to depend strongly on the nature of the polar headgroup of the lipid. The incorporation of cholesterol into glycerolmonooleate membranes reduced k_R, k_MS and k_S up to seven-fold.     

Modeling the membrane potential generation of bacteriorhodopsin

The archaeon Halobacterium salinarum can grow phototrophically with only light as its energy source. It uses the retinal containing and light-driven proton pump bacteriorhodopsin to enhance the membrane potential which drives the ATP synthase. Therefore, a model of the membrane potential generation of bacteriorhodopsin is of central importance to the development of a mathematical model of the bioenergetics of H. salinarum. To measure the current produced by bacteriorhodopsin at different light intensities and clamped voltages, we expressed the gene in Xenopus laevis oocytes. We present current-voltage measurements and a mathematical model of the current-voltage relationship of bacteriorhodopsin and its generation of the membrane potential. The model consists of three intermediate states, the BR, L, and M states, and comparisons between model predictions and experimental data show that the L to M reaction must be inhibited by the membrane potential. The model is not able to fit the current-voltage measurements when only the M to BR phase is membrane potential dependent, while it is able to do so when either only the L to M reaction or both reactions (L to M and M to BR) are membrane potential dependent. We also show that a decay term is necessary for modeling the rate of change of the membrane potential.     

Characterization of multi-layered membrane generated by rabies virus

Electron microscopy revealed multi-layered membranes within the cytoplasmic inclusion (accumulation of nucleocapsids) produced by rabies virus. When infected BHK cells were maintained at 31 C, an enhancement in production of these membranes occurred in approximately 60% of inclusion-containing cells. Multi-layered membranes were composed of an alternate array of two different layers; an electron-dense, thin membrane and a less dense layer which was thicker. SDS-polyacrylamide gel electrophoresis and immune electron microscopy of isolated multi-layered membrane preparations demonstrated that the structures contained viral G and M2 polypeptides. Our observations suggest that these membranous structures are not a degenerative product of rabies virus infection but rather are related to the replication of viral envelope constituents, although they represent themselves to be an abortive form of viral assembly.     

Stability of the two-dimensional lattice of bacteriorhodopsin reconstituted in partially fluorinated phosphatidylcholine bilayers

This study aims to investigate bacteriorhodopsin (bR) molecules reconstituted in lipid bilayers composed of di(nonafluorotetradecanoyl)-phosphatidylcholine (F4-DMPC), a partially fluorinated analogue of dimyristoyl-phosphatidylcholine (DMPC) to clarify the effects of partially fluorinated hydrophobic chains of lipids on protein's stability. Calorimetry measurements showed that the chain-melting transition of F4-DMPC/bR systems occurs at 3.5 °C, whereas visible circular dichroism (CD) and X-ray diffraction measurements showed that a two-dimensional (2D) hexagonal lattice formed by bR trimers in F4-DMPC bilayers remains intact even above 30 °C, similar to bR in a native purple membrane. Complete dissociation of the trimers into the monomers detected by visible CD almost coincides with the complete melting of 2D lattice observed by X-ray diffraction, in which both take place at around 65 °C (10 °C lower than that for bR in a native purple membrane). However, it is extremely high in comparison with the bR reconstituted in DMPC bilayers in which the dissociation of bR trimer in DMPC bilayers occurs near the chain-melting transition temperature of DMPC bilayers at approximately 18 °C. In order to explore the rationale behind the difference in stability, a further investigation of the detailed structural features of pure F4-DMPC bilayers was performed by analyzing the lamellar diffraction data using simple electron density models. The results suggested that the perfluoroalkyl groups do not exhibit any conformation change even if the chain-melting transition occurs, which is likely to contribute to the stability of the 2D hexagonal lattice formed by the bR trimers.     

Temperature dependence of water self-diffusion through lipid bilayers assessed by NMR

The temperature dependence of the coefficient of water self-diffusion across plane-parallel multib-ilayers of dioleoylphosphatidylcholine oriented on a glass support was studied in the 20–60°C range by pulsed field gradient NMR. The coefficient for transbilayer diffusion of water proved almost four orders of magnitude smaller than for bulk water, and 10 times smaller than that for lateral diffusion of lipid under the same conditions. The temperature dependence obeyed the Arrhenius law with apparent activation energy of 41 kJ/mol, much higher than that for bulk water (18 kJ/mol). The experimental data were analyzed using the “dissolution-diffusion” model, by simulating water passage through membrane channels, and by examining water exchange in states with different modes of translational mobility, including pore channels and bilayer defects. Each approach could take into account the role of bilayer permeability and assess the apparent activation energy for water diffusion in the hydrophobic part of the bilayer, which proved close to the value for bulk water. Estimates were obtained for water diffusion coefficients in the system, coefficients of bilayer permeability for water, and the influence of bilayer defects on the lateral and transverse diffusion coefficients.     

Photoinduced transformations in bacteriorhodopsin membrane monitored with optical microcavities

Photoinduced molecular transformations in a self-assembled bacteriorhodopsin (bR) monolayer are monitored by observing shifts in the near-infrared resonant wavelengths of linearly polarized modes circulating in a microsphere cavity. We quantify the molecular polarizability change upon all-trans to 13-cis isomerization and deprotonation of the chromophore retinal ( approximately -57 A(3)) and determine its orientation relative to the bR membrane ( approximately 61 degrees ). Our observations establish optical microcavities as a sensitive off-resonant spectroscopic tool for probing conformations and orientations of molecular self-assemblies and for measuring changes of molecular polarizability at optical frequencies. We provide a general estimate of the sensitivity of the technique and discuss possible applications.     

Probing the energy landscape of the membrane protein bacteriorhodopsin

The folding and stability of transmembrane proteins is a fundamental and unsolved biological problem. Here, single bacteriorhodopsin molecules were mechanically unfolded from native purple membranes using atomic force microscopy and force spectroscopy. The energy landscape of individual transmembrane alpha helices and polypeptide loops was mapped by monitoring the pulling speed dependence of the unfolding forces and applying Monte Carlo simulations. Single helices formed independently stable units stabilized by a single potential barrier. Mechanical unfolding of the helices was triggered by 3.9-7.7 A extension, while natural unfolding rates were of the order of 10(-3) s(-1). Besides acting as individually stable units, helices associated pairwise, establishing a collective potential barrier. The unfolding pathways of individual proteins reflect distinct pulling speed-dependent unfolding routes in their energy landscapes. These observations support the two-stage model of membrane protein folding in which alpha helices insert into the membrane as stable units and then assemble into the functional protein.