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.     

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.     

The photocycle of bacteriorhodopsin immobilized in poly(vinyl alcohol) film

The kinetics of photoelectric and optical signals were measured on samples containing oriented purple membranes immobilized in a poly(vinyl alcohol) film and on purple membranes introduced into a PVA-H2O mixture. The bacteriorhodopsin photocycle in the PVA-H2O mixture was complete. The only observed changes were the slowing down of the optical and electrical signals in relation to the M412-O640 and O640-bRall-trans steps. In the PVA film the O640 intermediate disappeared and a negative photoelectric signal appeared.     

Photochemically induced charge separation occurring in bacteriorhodopsin. Detection by time-resolved dielectric loss.

Time-resolved dielectric loss (TRDL) measurements are reported for the photochemical excitation of bacteriorhodopsin (bR) in solid films of Halobacterium halobium purple membranes. These measurements provide an independent confirmation for the existence of an important component of charge separation in these membranes after photochemical excitation. The separation of charge is detected by the absorption of microwave energy by the multilayer films of purple membranes in a microwave cavity during flash photolysis experiments. The TRDL method has the advantage of being sensitive to charge separation occurring in both oriented and unoriented films of purple membranes. One disadvantage is that the water content of the samples must be minimized, however, there is some absorbed water present in our electrodeposited solid film samples. To the best of our knowledge, TRDL measurements have not been reported previously for photochemical charge separation in biological membranes. It is significant that an early decay component of TRDL in the 20-microseconds time domain corresponds to the relaxation of the negative charge displacement photocurrent in oriented samples of purple membranes. In addition, a component of charge separation persists during the first several hundred microseconds of the bR photocycle.     

Photocurrent measurements of the purple membrane oriented in a polyacrylamide gel.

When illuminated, oriented purple membranes isolated from Halobacterium halobium give a photoelectric effect. The frequency response of a photocurrent measuring system for purple membranes oriented and immobilized in a polyacrylamide gel is analyzed from DC to 100 MHz. The waveform of the photocurrent can depend on both the sample conditions (including bathing solution) and the measuring system (electrode and ammeter) at both the low and high frequency ends. In the DC-1 kHz range (millisecond signals), the apparent lifetime of the photocurrent component is distorted if the electrode is not platinized and if the conductivity of the bathing solution is not low. In the 1 kHz to 1 MHz range (microsecond signals), the frequency response is flat under most conditions. In the MHz range (nanosecond signals), the apparent lifetime of the photocurrent component will be distorted if the conductivity of the bathing solution is not high and if the input impedance of the ammeter is not low and constant throughout the frequency range. With our optimized apparatus, we could measure the photocurrent components from oriented purple membrane with lifetimes from 70 ms to 32 ns without distortion by the measuring system.     

Influence of stray capacitance and sample resistance on the kinetics of fast photovoltages from oriented purple membranes

Flash-induced photovoltages were measured with metal electrodes in two experimental systems of purple membranes oriented by an electric field. One system consisted of a suspension of purple membranes cooled to 80 K. The photovoltage evoked by a xenon flash lamp displayed a single phase with a fast rise and a slow RC-decay. The signal shape is consistent with a fast charge separation occurring before the decay of the K-intermediate. The other system consisted of purple membranes embedded and stabilized in polyacrylamide gel. At room temperature, the photovoltage, evoked by a 10 ns laser flash, displayed a negative phase in the submicrosecond range and a slower positive one. The shape of the signals were altered in a complex manner by the stray capacitance and the ionic strength. The rise-time of the negative phase was approx. 14 and approx. 40 ns at ionic strengths of 10 and 1 mM, respectively. The initial peak amplitudes of the photovoltage from both experimental systems depended on the external capacitance in an inverse manner, indicating that both experimental systems were not impedance-matched. The evaluation of kinetic data of molecular reactions from measurements of the photovoltage is discussed.     

Orientation of intrinsic proteins in photosynthetic membranes. Polarized infrared spectroscopy of chloroplasts and chromatophores

In order to estimate the degree of orientation of the α-helices of intrinsic proteins in photosynthetic membranes, polarized infrared spectroscopy has been used to measure the dichroism of the amide I and amide II absorption bands of air-dried oriented samples of purple membranes, chloroplasts and chromatophores from Rhodopseudomonas sphaeroides. Using purple membrane, in which the orientation of the α-helices is precisely known (Henderson, R. (1977) Annu. Rev. Biophys. Bioeng. 6, 87–109), as a standard to calibrate our measurements and estimating the mosaic spread (extent of orientation) of the membranes from linear dichroism measurements performed in the visible spectral range, it is concluded that in photosynthetic membranes, the α-helices of intrinsic proteins are tilted at less than 40° with respect to the normal to the plane of the membrane.     

Elementary Energy Transfer Pathways in Allochromatium vinosum Photosynthetic Membranes

Allochromatium vinosum (formerly Chromatium vinosum) purple bacteria are known to adapt their light-harvesting strategy during growth according to environmental factors such as temperature and average light intensity. Under low light illumination or low ambient temperature conditions, most of the LH2 complexes in the photosynthetic membranes form a B820 exciton with reduced spectral overlap with LH1. To elucidate the reason for this light and temperature adaptation of the LH2 electronic structure, we performed broadband femtosecond transient absorption spectroscopy as a function of excitation wavelength in A. vinosum membranes. A target analysis of the acquired data yielded individual rate constants for all relevant elementary energy transfer (ET) processes. We found that the ET dynamics in high-light-grown membranes was well described by a homogeneous model, with forward and backward rate constants independent of the pump wavelength. Thus, the overall B800→B850→B890→ Reaction Center ET cascade is well described by simple triexponential kinetics. In the low-light-grown membranes, we found that the elementary backward transfer rate constant from B890 to B820 was strongly reduced compared with the corresponding constant from B890 to B850 in high-light-grown samples. The ET dynamics of low-light-grown membranes was strongly dependent on the pump wavelength, clearly showing that the excitation memory is not lost throughout the exciton lifetime. The observed pump energy dependence of the forward and backward ET rate constants suggests exciton diffusion via B850→ B850 transfer steps, making the overall ET dynamics nonexponential. Our results show that disorder plays a crucial role in our understanding of low-light adaptation in A. vinosum.     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Purple-to-blue transition of bacteriorhodopsin in a neutral lipid environment.

The red shift in the absorption maximum of native purple membrane suspensions caused by deionization is missing in lipid-depleted purple membrane, and the pK of the acid-induced transition is down-shifted to pH approximately 1.4 and has become independent of cation concentration (Szundi, I., and W. Stoeckenius. 1987. Proc. Natl. Acad. Sci. USA. 84:3681-3684). However, the proton pumping function cannot be demonstrated in these membranes. When native acidic lipids of purple membrane are exchanged for egg phosphatidylcholine or digalactosyldiglyceride, bacteriorhodopsin is functionally active in the modified membrane. It shows spectral shifts upon light-dark adaptation, a photocycle with M-intermediate and complex decay kinetics; when reconstituted into vesicles with the same neutral lipids, it pumps protons. Unlike native purple membrane, lipid-substituted modified membranes do not show a shift of the absorption maximum to longer wavelength upon deionization. A partial shift can be induced by titration with HCl; it has a pK near 1.5 and no significant salt dependence. Titration with HNO3 and H2SO4, which causes a complete transition in the lipid-depleted membranes, i.e., it changes their colors from purple to blue, does not cause the complete transition in the lipid-substituted preparations. These results show that the purple color of bacteriorhodopsin is independent of cations and their role in the purple-to-blue transition of native membranes is indirect. The purple and blue colors of bacteriorhodopsin are interpreted as two conformational states of the protein, rather than different protonation states of a counterion to the protonated Schiff base.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field-induced absorption changes of bacteriorhodopsin were studied with different samples of purple membranes which were prepared as randomly oriented and electrically oriented films of purple as well as cation-depleted blue bacteriorhodopsin. The absorption changes were proportional to the square of the field strength up to 300 kV/cm. The electric field from the intracellular side to the extracellular side of the purple bacteriorhodopsin induces a spectrum change, resulting in a spectrum similar to that of the cation-depleted blue bacteriorhodopsin. When the field was removed, the purple state was regenerated. The blue state was mainly affected by an electric field in the opposite direction, suggesting a reversible interaction with the Schiff's base bond of the retinal. Since the field-induced reaction of bacteriorhodopsin was observed in the presence of a concomitant steady ion flux, it is assumed that the generation of a local diffusion potential may play an important role in these spectral reactions. Although the fragments were fixed in the dried film, electric dichroism was observed. The dichroic contribution of the total absorbance change was about 15%. The angular displacement of the retinal transition moment was calculated to be 1.5° toward the membrane normal.     

Comparative studies on the fine structure of purple membrane fromHalobacterium cutirubrum andHalobacterium halobium

Summary Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane fromHalobacterium cutirubrum andHalobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane fromHalobacterium halobium, it must be similarly so for the membrane fromHalobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.     

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halobium

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.     

Charge motions during the photocycle of pharaonis halorhodopsin

Oriented gel samples were prepared from halorhodopsin-containing membranes from Natronobacterium pharaonis, and their photoelectric responses to laser flash excitation were measured at different chloride concentrations. The fast component of the current signal displayed a characteristic dependency on chloride concentration, and could be interpreted as a sum of two signals that correspond to the responses at high-chloride and no-chloride, but high-sulfate, concentration. The chloride concentration-dependent transition between the two signals followed the titration curve determined earlier from spectroscopic titration. The voltage signal was very similar to that reported by another group (Kalaidzidis, I. V., Y. L. Kalaidzidis, and A. D. Kaulen. 1998. FEBS Lett. 427:59-63). The absorption kinetics, measured at four wavelengths, fit the kinetic model we had proposed earlier. The calculated time-dependent concentrations of the intermediates were used to fit the voltage signal. Although no negative electric signal was observed at high chloride concentration, the calculated electrogenicity of the K intermediate was negative, and very similar to that of bacteriorhodopsin. The late photocycle intermediates (O, HR', and HR) had almost equal electrogenicities, explaining why no chloride-dependent time constant was identified earlier by Kalaidzidis et al. The calculated electrogenicities, and the spectroscopic information for the chloride release and uptake steps of the photocycle, suggest a mechanism for the chloride-translocation process in this pump.     

Electric field induced conformational changes of bacteriorhodopsin in purple membrane films

Electric field induced conformational changes of bacteriorhodopsin were studied in six types of dried film (randomly and electrically oriented membranes of purple as well as cation-depleted blue bacteriorhodopsin) by measuring the frequency dependence of the optical absorbance change and the dielectric dispersion and absorption. For the purple bacteriorhodopsin the optical absorbance change induced by alternating rectangular electric fields of ±300 kV/cm altered the sign twice in the frequency range from 0.001 Hz to 100 kHz (around 0.03 Hz and 100 kHz), indicating that the electric field induced conformational change in these samples consists of, at least, three steps. Similarly, it was found for the blue bacteriorhodopsin that at least two steps are involved. In accord with optical measurements, the dielectric behaviour due to alternating sinusoidal electric fields of±6kV/cm in the frequency range from 10 Hz to 10 MHz showed two broad dispersion/absorption regions, one below 1 kHz and the other around 10–100 kHz. This suggests that the conformational change of bacteriorhodopsin is also reflected by its dielectrical properties and that it is partially induced at 6 kV/cm. Including previous results obtained by analysis of the action of DC fields on purple membrane films, a model for a field-induced cyclic reaction for purple as well as blue bacteriorhodopsin is proposed. In addition it was found that there are electrical interactions among purple membrane fragments in dried films.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Genetic cloning and functional expression in Escherichia coli of an archaerhodopsin gene from Halorubrum xinjiangense

Pairs of PCR primers that targeted the archae/bacteriorhodopsin gene were used to clone the archaerhodopsin (aR) gene of Halorubrum xinjiangense strain BD-1^T, and this gene was sequenced and functionally expressed in Escherichia coli. Recombinant E. coli cells harboring the plasmid carrying this gene became slightly purple or blue depending on whether they were supplemented with all- trans retinal or 3,4-dihydroretinal, respectively, during induction with IPTG. The purple and blue membranes from the recombinant E. coli showed maximal absorption at 555 and 588 nm, respectively, which are different from maximal absorption at 568 nm of the wild-type purple membrane. Purple membranes from the recombinant E. coli and from strain BD-1^T were investigated in parallel. The E. coli purple membrane was fabricated into films and photoelectric responses were observed that depended on the light-on and light-off stimuli.     

Stability of transmembrane regions in bacteriorhodopsin studied by progressive proteolysis

Summary Proteinase K digestions of bacteriorhodopsin were carried out with the aim of characterizing the membrane-embedded regions of the protein. Products of digestions for two, eight or 24 hours were separated by high-pressure liquid chromotography. A computerized search procedure was used to compare the amino acid analyses of peptide-containing peaks with segments of the bacteriorhodopsin sequence. Molecular weight distributions of the products were determined by sodium dodecylsulfate-urea polyacrylamide gel electrophoresis. The structural integrity of the protein after digestion was monitored through the visible absorption spectrum, by X-ray diffraction of partially dried membranes, and by following release of biosynthetically-incorporated³H leucine from the digested membranes.During mild proteolysis, bacteriorhodopsin was cleaved near the amino and carboxyl termini and at two internal regions previously identified as being accessible to the aqueous medium. Longer digestion resulted in cleavage at new sites. Under conditions where no fragments of bacteriorhodopsin larger than 9000 mol wt were observed, a significant proportion of the digested membranes retained diffraction patterns similar to those of native purple membranes. The harshest digestion conditions led to complete loss of the X-ray diffraction patterns and optical absorption and to release of half the hydrophobic segments of the protein from the membrane in the form of small soluble peptides. Upon cleavage of aqueous loop regions of the protein, isolated transmembrane segments may experience motion in a direction perpendicular to the plane of the membrane, allowing them access to protease.     

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.     

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.