Purple Membrane Fragments Research Articles

Photogenerated movement of protons through bacteriorhodopsin in relation to the analysis of photoelectrical responses

Bacteriorhodopsin, a protein–retinal complex, forms two-dimensional patches located on the membrane of a photosynthetic bacterium. These patches, called purple-membrane fragments, act as light-driven proton pumps converting the light energy into a transmembrane gradient of proton electrochemical potential.Purple-membrane fragments are adsorbed on the surface of bimolecular lipid membranes doped with a lipophilic proton carrier and separating two aqueous solutions of controlled pH. We have measured the photocurrents associated with the photogenerated proton movements through the bacteriorhodopsin. In particular the influence of the light energy, the pH of the aqueous media and the applied voltage have been investigated, the main result being a reversal in the direction of proton pumping.In order to account for these results, a model of phototransport of protons through the bacteriorhodopsin is proposed. According to the model, the pathway for proton movement is composed of two chains of amino acids acting as proton-conducting wires which connect the aqueous solutions to sites able to exchange protons with the photoactive heart. Undoubtedly the photoactive heart contains the Schiff base of the retinal. From this transport model it appears that the degree of protonation of the donor sites which exchange protons with the photoactive heart plays a central role in the working of the pump.A kinetic expression of the stationary photocurrent is derived from the model and accounts for the experimental data.

LIGHT‐INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light‐induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR570) and M412‐intermediate, but decreased the decay rate of the M412‐intermediate. Light‐induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light‐induced proton transport. The role of amino group(s) in light‐induced proton release and transport through the purple membrane is discussed.

Variable temperature photoacoustic spectroscopy. II. Temperature characteristic and applications

The temperature dependent photoacoustic signal of a carbon black sample is investigated with a previously described low temperature photoacoustic cell. Examples are given to demonstrate the utility of variable temperature photoacoustic spectroscopy: The photocycle of bacteriorhodopsin which is contained in the purple membrane fragments of halobacterium halobium is studied. In this experiment some photointermediates are shown to be preferentially populated and frozen out at low temperatures. The temperature dependence of the electronic ground state of solid Fe-(III)-diethyl-dithiocarbamate is investigated for the first time spectroscopically in the visible and ultraviolet spectral range. Excitonic levels in semiconductors are for the first time inspected by the photoacoustic technique.

Photocurrent kinetics of purple-membrane sheets bound to planar bilayer membranes

The kinetics of light-driven proton transport by bacteriorhodopsin has been studied in a model system consisting of a planar lipid bilayer membrane to which purple membrane fragments have been attached. After excitation with a 10-nsec laser flash a fast negative current-transient occurs, followed by a positive transient which decays to zero. The time course of the photocurrent may be represented by a sum of four exponentials with time constantsτ1= 1.2μsec,τ2= 17μsec,τ4= 57μsec,τ1= 950μsec (at 25°C). In a D2O mediumτ2 andτ3 are increased by a factor of 2.6 and 2.9, respectively, whereasτ1 remains unaffected. The observed components of the photocurrent can be correlated to photochemical reaction steps inferred from flash-photometric experiments on the basis of the observed time constants, the activation energies, and the effects of pH and D2O. From the photocurrent signals information may be obtained on the magnitude of the charge displacement associated with the elementary transitions of the bacteriorhodopsin molecule.

Photoacoustic calorimetry of Halobacterium halobium photocycle

Enthalpy changes that occur during the photocycle of Halobacterium halobium purple membrane fragments have been measured on a millisecond time scale, using photoacoustic detection to obtain modulation photocalorimetric data. Details are given on the ways to obtain quantitative thermodynamic and kinetic information by this method.

334 - Electrochemical analysis of transmembrane proton fluxes photogenerated by bacteriorhodopsin in B.L.M.

The variation with time of photocurrents generated by bacteriorhodopsin in the form of purple membrane fragments (PMF) incorporated in a B.L.M. are recorded. These generally increase at first and then decrease down to positive or negative values. The shape of the curves depend on the light energy, the value of the transmembrane electrical field and the pH of the aqueous solutions. The results are interpreted both on the basis of an initial active transport of protons across the purple membrane fragments as a result of the illumination and passive fluxes of part of the protons which have been transported. A general treatment is proposed to account for the experimental results based on an application of electrochemical kinetics to the proton transfer.

Surface charge changes in purple membranes and the photoreaction cycle of bacteriorhodopsin.

The surface potential of purple membrane fragments, determined from the distribution of the aqueous free and the membrane-bound positively charged, paramagnetic, amphiphilic probe 4-(dodecyldimethylammonium)-1-oxyl-2,2,6,6-tetramethylpiperidine bromide varied almost 60 mV as a function of ionic strength and 50 mV as a function of pH of the medium. Light-induced changes in surface potential followed the changes observed in the M412 intermediate of the photocycle of bacteriorhodopsin as a function of pH, temperature, and response to antibiotics beauvericin and valinomycin. The number of induced charges per M412 appearing at the surface of purple membranes decreased from about 0.75 to 0.45 as the surface potential became more negative. The stoichiometry would be twice as large if the charge changes were localized exclusively on one side of the purple membrane. Laser flash-induced kinetics of the rise and decay of surface charge changes were slightly slower than the kinetics of the rise and decay of M412 which is associated with the reversible deprotonation of the retinal Schiff base nitrogen in the chromophore. It is suggested that the light-induced charge changes monitor a dissociable amino acid residue which may be a step in the movement of protons across the purple membrane.

Surface-induced lamellar orientation of multilayer membrane arrays. Theoretical analysis and a new method with application to purple membrane fragments

The orientation of membrane fragments into a lamellar array by a flat surface is analyzed. This analysis includes processes such as centrifugation and drying and physical effects due to membrane fragment steric interactions, finite size, elasticity, and thermal fluctuations. Several model calculations of optimal orientational order in multilayer membrane arrays are presented. The predictions of a smectic A model agree quantitatively with the measured spatial dependence of the fluctuations in layer orientation in a multilamellar arrays. A new technique, based in part on this analysis, for the preparation of well-oriented multilamellar arrays of natural and artificial membranes, isopotential spin-dry centrifugation, is described. The method involves the use of specially designed inserts for the buckets of a standard vacuum ultracentrifuge. The membrane fragments to be oriented are sedimented from solution or suspension onto a substrate of a convenient material which forms a gravitational isopotential surface at high g. Sedimentation is accompanied by removal of the suspending medium at high g to produce oriented films with a selected degree of solvation. In addition, a method is described whereby small solute molecules can be maintained in constant concentration with the membrane fragments during this process. Initial application of the method to the orientation of purple membrane fragments is described. The degree of orientation obtained in this system is evaluated using freeze-fracture and scanning electron microscopy, optical birefringence, linear dichroism, and microscopy.

Orientation of membrane fragments by electric field

Purple membrane fragments from Halobacterium halobium were oriented by a static electric field in a water suspension. It was found that an electric field of approx. 20 V/cm is sufficient to achieve practically complete orientation; the purple membranes have a permanent electric dipole moment of (6 ±1)· 10 −23 C · m, the orientation of the retinal transition moment relative to the direction of the electric dipole moment, θ, is (59 ± 1) 0, and the purple membrane rotational diffusion constant D rot = 0.65 s −1. It was found that because of the electrophoretic movement of the particles a hydrodynamic velocity gradient builds up which also orients the purple membranes.

Photocurrent response of bacteriorhodopsin adsorbed on bimolecular lipid membranes

The photo response of bacteriorhodopsin adsorbed on a bimolecular lipid membrane has been investigated using short-circuit current measurements. The results revealed a biphasic current vs. time curve for the photocurrent at pH values of approx. 7. This phenomenon could be modified by altering either the value of the external applied electrical field or the proton concentration differences. The observed effects of the external applied voltage, pH gradient and lipophilic proton carriers enabled us to conclude that the bacteriorhodopsin can be adsorbed in two different states, which give rise to a pumping effect and a flux of protons in opposite directions. A theoretical analysis of the photocycle in relation to the electrical field which acts on the proton uptake and release is proposed. The main effect of this field is to diminish the pumping rate due to the proton motive force resulting from the creation of space-charge in the vicinity of purple membrane fragments.

Photoelectrospectrometry of bilayer lipid membranes

Three different bilayer lipid membrane systems were studied under visible and ultraviolet illumination. The first system consisted of a bilayer lipid membrane formed with a mixture of phospholipids and cholesterol, to one side of which purple membrane fragments from Halobacterium halobium were added. The second system consisted of a membrane formed from spinach chloroplast extract. When either of these membrane systems was illuminated with ultraviolet and visible radiation, photopotentials were observed and photoelectric action spectra were recorded (the technique is termed photoelectrospectrometry). Each spectrum had a definite structure which was characteristic of each of the modified membranes. The third system studied consisted of an otherwise photoinactive membrane formed with a mixture of phospholipids and cholesterol, to one side of which chymotrypsin was added. When the membrane was illuminated with visible light no photoresponse was observed. On the other hand, a photopotential which increased with incubation time was observed when the membrane was illuminated with ultraviolet light. Since, in our systems, the photoresponses have been observed to be due to certain species incorporated into the membrane, it appears that photoelectrospectrometry is a useful tool for studying lipid-protein interactions, constituent organization and energy transfer in membranes.

LIGHT AND pH INDUCED CHANGES IN THE SURFACE PRESSURE‐AREA ISOTHERMS OF BACTERIORHODOPSIN

Abstract— Pressure‐area isotherms of monolayers of purple membrane fragments were measured at an air‐water interface. The pH of the aqueous phase was varied from 2 to 10. Over the pH range 5 to 9 the area/molecule of bacteriorhodopsin was constant and illumination of the film had no effect on the isotherms.At pH's more acid than 5 and more alkaline than 9, there were large decreases in area/molecule. Under these conditions of extreme pH the monolayer was very sensitive to light. Illumination resulted in irreversible increases in area/molecule.

Electrochemical analysis of transmembrane proton fluexes photogenerated by bacteriorhodopsin in B.L.M.

The variation with time of photocurrents generated by bacteriorhodopsin in the form of purple membrane fragments (PMF) incorporatated in a B.L.M. are recorded. These generally increase at first and then decrease down to positive or negative values. The shape of the curves depend on the light energy, the value of the transmembrane electrical field and the pH of the aqueous solutions. The results are interpreted both on the basis of an initial active transport of protons across the purple membrane fragments as a result of the illumination and passive fluxes of part of the protons which have been transported. A general treatment is proposed to account for the experimental results based on an application of electrochemical kinetics to the proton transfer.

Resonance Raman evidence for secondary protein—Schiff base interactions in bacteriorhodopsin: correlation of the primary excitation mechanism with a model for proton pumping and visual transduction

Resonance Raman spectra of bacteriorhodopsin (in various isotopically labelled and natural environments) and rhodopsins demonstrate that in all bacteriorhodopsin species with protonated Schiff base linkages, there is a secondary protein-Schiff base interaction. Steady state resonance Raman spectra of 15 N labelled purple membrane fragments and kinetic resonance Raman spectra (k.r.R.s.) as a function of pH, are consistent with the suggestion that the e-amino group of a lysine residue is responsible for this secondary protein-protonated Schiff base interaction. Spectra of bacteriorhodopsin photo-intermediates with unprotonated Schiff base linkages demonstrate that the secondary protein-Schiff base interaction is absent in these species. These results, together with the accessibility of the protonated Schiff base in bacteriorhodopsin (bR 570 ) to solvent from the external medium and the insensitivity to pH of the secondary lysine-Schiff base complex in bR 570 , suggest a structure for the complex in which the Schiff base proton is interacting with an amino acid side chain in an unprotonated configuration. In addition k.r.R.s. as a function of pH has demonstrated that the protein-Schiff base complex has a p K > 12 before light absorption and a p K in the range between 9.9 and 10.3 microseconds after light absorpion. These results suggest a molecular mechanism for proton pumping, which appears to account for the changes that are observed in the visible absorption spectrum and the resonance Raman spectrum as a function of proton pumping and pH. Finally, our results on bacteriorhodopsin and rhodopsin indicate that the primary excitation mechanism in these pigments, which produce remarkably similar absorption red shifts on similar time scales with different chromophore conformations and Schiff base interactions, must involve some region in the active site removed from the Schiff base and unrelated to a simple 11- cis to all- trans isomerization. A proton translocation in the protein together with retinal structural alteration (Lewis 1978) certainly fits all our observations and results in plausible molecular transformations which can account for the spectral similarities of all rhodopsins and bacteriorhodopsin while accounting for their functional diversity.

284 - H. Halobium : I. In vitro studies

1. Purple membrane fragments (PM) from H. halobium were incorporated into lipid membranes either directly or via liposomes. A photoresponse was detected when liposomes containing PM were fused with lipid membranes. Low—resistance membranes or membranes shunted with an external resistor of 10 9 ohms showed decay of the initial light response to some equilibrium value in the light in both presence and absence of octadecylamine. The light response could be abolished by the addition of a sufficient amount of triethylamine to either side of the membrane. 2. The photovoltage action spectra of bilayer lipid membranes containing PM either directly or via liposomes were measured, and found to follow the absorption spectrum of bacteriorhodopsin. 3. Liposomes containing bacteriorhodopsin (BR) in PM extract of H. halobium were fused to one side of planar lipid membranes. The photopotential resulting from flash excitation rose and fell as the sum of three exponentials with time constants for the leading edge of 30 ± 10 μs and 35 ± 10 ms. The decay time constant of the photopotential was 840 ms, a value consistent with the membrane time constant given by the membrane dark resistance and capacitance.

BRANCHING IN THE BACTERIORHODOPSIN PHOTOCHEMICAL CYCLE

Abstract— Kinetics have been determined for the decay of the terminal phototransients M(410) and O(660) and the reappearance of the BR(570) chromophore in flash‐photolyzed aqueous suspensions of light‐adapted purple membrane fragments from Halobacterium halobium. The results were fitted to a linear model of

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.

Interaction of purple membrane with solvents. I. Applicability of solubility parameter mapping

We carried out spectral studies on the interaction between purple membrane fragments (isolated from Halobacterium halobium) and a series of different solvents, classified quantitatively according to their solubility parameters δ d , δ p , δ h . These represent the contribution of dispersion forces, polar forces, and hydrogen bonding, respectively, to the cohesive energy density of the solvent. Purple membrane fragments, kept in the dark, were suspended in each of the solvents as well as in binary mixtures of solvents, and the spectrum of the resulting suspension was recorded in the wavelength region 250–700 nm. The interaction of each solvent with the membrane fragments can be represented by a point on either a ternary diagram, where each of the three axes represents one of the solubility parameters, or a binary diagram, where one of the two axes is a combination of two of the solubility parameters ( δ v = δ d 2 + δ p 2 or δ p 2 + δ h 2 ). In the former type of solvent map the contribution of each of the parameters is distinct but only their relative contributions are expressed. In the latter the absolute values of δ i are considered. In each of these modes of presentation an inner closed region is observed. The solvents inside its borders interact with bacteriorhodopsin with a resultant spectral change. Mixtures of solvents fit the maps according to their calculated δ values. Thus, a mixture of an apolar solvent with a highly polar solvent interacts with bacteriorhodopsin, even though each of these solvents alone does not.

The quantum yield of flash-induced proton release by bacteriorhodopsin-containing membrane fragments

The quantum yield of proton release by bacteriorhodopsin was measured from volume changes after excitation of purple membrane fragments by short flashes. At low ionic strengths, about 0.25 mol of protons is released per einstein absorbed. This agrees well with quantum yields reported recently for the conversion of bacteriorhodopsin into a metastable state (M) that absorbs near 412 nm. However, the quantum yield of proton release increases gradually with increasing ionic strength; it plateaus with a value of 0.43 +/- 0.03 at ionic strengths above 200 mM. Changing the ionic strength has no detectable effect on the quantum yield of formation of the M spectral state. It thus appears that as many as two protons can be released and rebound in each photochemical cycle at high ionic strengths. The quantum yield of proton release is essentially independent of pH over the range 6.0-8.75. The quantum yield decreases with increasing flash strength, apparently due to photoreversal of the initial photochemical reaction.

H. Halobium: I. In vitro studies

Summary o 1. Purple membrane fragments (PM) from H. halobium were incorporated into lipid membranes either directly or via liposomes. A photoresponse was detected when liposomes containing PM were fused with lipid membranes. Low-resistance membranes or membranes shunted with an external resistor of 109 ohms showed decay of the initial light response to some equilibrium value in the light in both presence and absence of octa decylamine. The light response could be abolished by the addition of a sufficient amount of triethylamine to either side of the membrane. 2. The photovoltage action spectra of bilayer lipid membranes containing PM either directly or via liposomes were measured, and found to follow the absorption spectrum of bacteriorhodopsin. 3. Liposomes containing bacteriorhodopsin (BR) in PM extract ofH. halobium were fused to one side of planar lipid membranes. The photopotential resulting from flash excitation rose and fell as the sum of three exponentials with time constants for the leading edge of 30±10 μs and 35±10 ms. The decay time constant of the photopotential was 840 ms, a value consistent with the membrane time constant given by the membrane dark resistance and capacitance.