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.     

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.     

On the two forms of bacteriorhodopsin

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int. 30,461-469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (τ₁ = 60–70 μs and τ₂ = 220–250 μs), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the D115N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.0–5.5 only the M-intermediate with a rise time of 60 μs was present. A 5–6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bacteriorhodopsin contained 100% fast M-intermediate. The disappearance of the 250-μs phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.     

Structure of an early intermediate in the M-state phase of the bacteriorhodopsin photocycle.

The structure of an early M-intermediate of the wild-type bacteriorhodopsin photocycle formed by actinic illumination at 230 K has been determined by x-ray crystallography to a resolution of 2.0 A. Three-dimensional crystals were trapped by illuminating with actinic light at 230 K, followed by quenching in liquid nitrogen. Amide I, amide II, and other infrared absorption bands, recorded from single bacteriorhodopsin crystals, confirm that the M-substate formed represents a structure that occurs early after deprotonation of the Schiff base. Rotation about the retinal C13-C14 double bond appears to be complete, but a relatively large torsion angle of 26 degrees is still seen for the C14-C15 bond. The intramolecular stress associated with the isomerization of retinal and the subsequent deprotonation of the Schiff base generates numerous small but experimentally measurable structural changes within the protein. Many of the residues that are displaced during the formation of the late M (M(N)) substate formed by three-dimensional crystals of the D96N mutant (Luecke et al., 1999b) are positioned, in early M, between their resting-state locations and the ones which they will adopt at the end of the M phase. The relatively small magnitude of atomic displacements observed in this intermediate, and the well-defined positions adopted by nearly all of the atoms in the structure, may make the formation of this structure favorable to model (simulate) by molecular dynamics.     

Structural transition of bacteriorhodopsin is preceded by deprotonation of Schiff base: microsecond time-resolved x-ray diffraction study of purple membrane

The structural changes in the photoreaction cycle of bacteriorhodopsin, a light-driven proton pump, was investigated at a resolution of 7 angstroms by a time-resolved x-ray diffraction experiment utilizing synchrotron x rays from an undulator of SPring-8. The x-ray diffraction measurement system, used in coupling with a pulsed YAG laser, enabled us to record a diffraction pattern from purple membrane film at a time-resolution of 6 micros over the time domain of 5 micros to 500 ms. In the time domain, the functionally most important M-intermediate appears. A series of time-resolved x-ray diffraction data after photo-excitation showed clear intensity changes caused by the conformational changes of helix G in the M-intermediate. The population of the reaction intermediate was prominently observed at approximately 5 ms after a photo-stimulus. In contrast, absorption measurement indicated the deprotonation of the Schiff base predominantly occurred at approximately 300 micros after a photo-stimulus. These results showed that the conformational changes characterizing structurally the M-intermediate predominantly occur at a later stage of the deprotonation of the Schiff base. Thus, the M-intermediate can be divided into two metastable stages with different physical characteristics.     

Orientation of the protonated retinal Schiff base group in bacteriorhodopsin from absorption linear dichroism.

Linear dichroism experiments are performed on light-adapted bacteriorhodopsin (BR568) films containing native retinal (A1) and its 3,4-dehydroretinal (A2) analogue to measure the angle between the chromophore transition dipole moment and the membrane normal. QCFF/pi calculations show that the angle between the transition moment and the long axis of the polyene is changed by 3.4 degrees when the C3-C4 bond is unsaturated. The difference vector between the two transition moments points in the same direction as the Schiff base (N----H) bond for the all-trans BR568 chromophore. Because the plane of the chromophore is perpendicular to the membrane plane, a comparison of the transition moment orientations in the A1- and A2-pigments enables us to determine the orientation of the N----H bond with respect to the absolute chromophore (N----C5 vector) orientation. The angles of the transition moments are 70.3 degrees +/- 0.4 degrees and 67.8 degrees +/- 0.4 degrees for the A1- and A2-pigments, respectively. The fact that the change in the transition moment angle (2.5 degrees) is close to the predicted 3.4 degrees supports the idea that the chromophore plane is nearly perpendicular to the membrane plane. The decreased transition moment angle in the A2-analogue requires that the N----H bond and the N----C5 vector point toward the same membrane surface. Available results indicate that the N----C5 vector points toward the exterior in BR568. With this assignment, we conclude that the N----H bond points toward the exterior surface and its most likely counterion Asp-212. This information makes possible the construction of a computer graphics model for the active site in BR568.     

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.     

Two progressive substrates of the M-intermediate can be identified in glucose-embedded, wild-type bacteriorhodopsin.

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.     

Conformational changes in bacteriorhodopsin studied by infrared attenuated total reflection.

We report on a new method based on Fourier transform infrared (FTIR)-difference spectroscopy for studying the conformational changes occurring during the photocycle of bacteriorhodopsin. Previous studies have been made by measuring the absorbance of an infrared (IR) beam transmitted through a thin hydrated purple membrane film. In contrast, the present study utilizes the technique of attenuated total reflection (ATR). Purple membrane is fixed on the surface of a germanium internal reflection crystal and immersed in a buffer whose pH and ionic composition can be varied. Measurements of the amide I and II absorbance with light polarized parallel and at 45 degrees to the crystal surface reveals that the membrane is highly oriented. An ATR-FTIR-difference spectrum of the light to dark (bR570 to bR548) transition is similar but not identical to the transmittance FTIR-difference spectrum. This disagreement between the two methods is shown to be due in the ATR case to the absorption of transition moments oriented predominantly out of the membrane plane. Raising the pH of La3+ substituted purple membrane films from 6.8 to 8.0 slows the M-decay rate sufficiently so that a bR570 to M412 difference spectrum can be obtained with steady state illumination at room temperature. A comparison of this difference spectrum with that obtained at -23 degrees C using the transmittance method reveals several changes that cannot be attributed to out-of-plane transition moments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transient and linear dichroism studies on bacteriorhodopsin: determination of the orientation of the 568 nm all-trans retinal chromophore

The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane.     

Characterization of the conformational change in the M1 and M2 substates of bacteriorhodopsin by the combined use of visible and infrared spectroscopy.

A combination of visible and Fourier transform infrared (FTIR) spectroscopies is used to characterize the formation of the M1 and M2 substates of the bacteriorhodopsin photocycle in glucose-embedded, hydrated thin films. Difference FTIR bands in the amide I region verify the previously reported existence of a significant peptide backbone conformational change in the transition from M1 to M2. The visible absorption spectra demonstrate that contamination of the M-intermediate samples by L, N, or other non-M species should contribute negligibly to the observed changes in the amide I region, and this conclusion is supported by comparison of specific carboxyl group peaks with corresponding bands in published L and N FTIR difference spectra. Based upon spectroscopic results, an extension of the C-T Model (Fodor, S., Ames, J., Gebhard, R., van den Berg, E., Stoeckenius, W., Lugtenberg, J., and Mathies, R. (1988) Biochemistry 27, 7097-7101) is presented. The results of this work suggest that protein structural changes should be clearly visible in M-bR, difference Fourier density maps and that these structural changes may in turn elucidate how bacteriorhodopsin actively pumps ions across the purple membrane of Halobacterium halobium.     

Effect of high pressure on the absorption spectrum and isomeric composition of bacteriorhodopsin.

The effects of high pressure upon the absorption spectra and isomeric composition of the dark (bRD) and light adapted (bRL) forms of bacteriorhodopsin were examined. Pressure favors the 13-cis form of bacteriorhodopsin (bR13-cis). The equilibrium isomeric composition and absorption spectra of bacteriorhodopsin samples at a given pressure were the same starting from either light or dark adapted bacteriorhodopsin. From the effect of pressure on the equilibrium constant between bRall-trans in equilibrium bR13-cis in the dark, the molar volume change between bRall-trans and bR13-cis was found to be -7.8 +/- 3.2 ml/mol. This volume change suggests a difference in conformation between dark- and light-adapted bacteriorhodopsin, but the magnitude of the change is small, involving only a small number of the protein residues.     

Dielectric dispersions in bacteriorhodopsin

Dispersion effects in bacteriorhodopsin both in suspension and incorporated into liposomes have been studied by measuring the changes in the dielectric properties induced by electric and magnetic fields at low and medium frequencies. The samples exhibit very high values of relative permittivity and dielectric loss. Dispersions have been measured up to 200 kHz and are believed to be due to the reorientation of the bacteriorhodopsin chromophore within the membrane fragments. A study of relaxation times vs temperature shows a transition at 28°C, the same temperature as found using other techniques.     

Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study

Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.     

Crystallization of bacteriorhodopsin from bicelle formulations at room temperature

We showed previously that high-quality crystals of bacteriorhodopsin (bR) from Halobacterium salinarum can be obtained from bicelle-forming DMPC/CHAPSO mixtures at 37 degrees C. As many membrane proteins are not sufficiently stable for crystallization at this high temperature, we tested whether the bicelle method could be applied at a lower temperature. Here we show that bR can be crystallized at room temperature using two different bicelle-forming compositions: DMPC/CHAPSO and DTPC/CHAPSO. The DTPC/CHAPSO crystals grown at room temperature are essentially identical to the previous, twinned crystals: space group P21 with unit cell dimensions of a = 44.7 A, b = 108.7 A, c = 55.8 A, beta = 113.6 degrees . The room-temperature DMPC/CHAPSO crystals are untwinned, however, and belong to space group C222(1) with the following unit cell dimensions: a = 44.7 A, b = 102.5 A, c = 128.2 A. The bR protein packs into almost identical layers in the two crystal forms, but the layers stack differently. The new untwinned crystal form yielded clear density for a previously unresolved CHAPSO molecule inserted between protein subunits within the layers. The ability to grow crystals at room temperature significantly expands the applicability of bicelle crystallization.     

Compositional heterogeneity reflects partial dehydration in three-dimensional crystals of bacteriorhodopsin

Absorption, fluorescence and excitation spectra of three-dimensional bacteriorhodopsin crystals harvested from a lipidic cubic phase are presented. The combination of the spectroscopic experiments performed at room temperature, controlled pH and full external hydration reveals the presence of three distinct protein species. Besides the well-known form observed in purple membrane, we find two other species with a relative contribution of up to 30%. As the spectra are similar to those of dehydrated or deionized membranes containing bacteriorhodopsin, we suggest that amino acid residues, located in the vicinity of the retinal chromophore, have changed their protonation state. We propose partial dehydration during crystallization and/or room temperature conditions as the main source of this heterogeneity. This assignment is supported by an experiment showing interconversion of the species upon intentional dehydration and by crystallographic data, which have indicated an in-plane unit cell in 3D crystals comparable to that of dehydrated bacteriorhodopsin membranes. Full hydration of the proteins after the water-withdrawing crystallization process is hampered. We suggest that this hindered water diffusion originates mainly from a closure of hydrophobic crystal surfaces by lipid bilayers. The present spectroscopic work complements the crystallographic data, due to its ability to determine quantitatively compositional heterogeneity resulting from proteins in different protonation states.     

Transmembrane location of retinal in bacteriorhodopsin by neutron diffraction

The transmembrane location of the chromophore of bacteriorhodopsin was obtained by neutron diffraction on oriented stacks of purple membranes. Two selectively deuterated retinals were synthesized and incorporated in bacteriorhodopsin by using the retinal- mutant JW5: retinal-d11 (D11) contained 11 deuterons in the cyclohexene ring, and retinal-d5 (D5) had 5 deuterons as close as possible to the Schiff base end of the chromophore. The membrane stacks had a lamellar spacing of 53.1 A at 86% relative humidity. Five orders were observed in the lamellar diffraction pattern of the D11, D5, and nondeuterated reference samples. The reflections were phased by D2O-H2O exchange. The absolute values of the structure factors were nonlinear functions of the D2O content, suggesting that the coherently scattering domains consisted of asymmetric membrane stacks. The centers of deuteration were determined from the observed intensity differences between labeled and unlabeled samples by using model calculations and Fourier difference methods. With the origin of the coordinate system defined midway between consecutive intermembrane water layers, the coordinates of the center of deuteration of the D11 and D5 label are 10.5 +/- 1.2 and 3.8 +/- 1.5 A, respectively. Alternatively, the label distance may be measured from the nearest membrane surface as defined by the maximum in the neutron scattering length density at the water/membrane interface. With respect to this point, the D11 and D5 labels are located at a depth of 9.9 +/- 1.2 and 16.6 +/- 1.5 A, respectively. The chromophore is tilted with the Schiff base near the middle of the membrane and the ring closer to the membrane surface. The vector connecting the two label positions in the chromophore makes an angle of 40 +/- 12 degrees with the plane of the membrane. Of the two possible orientations of the plane of the chromophore, which is perpendicular to the membrane plane, only the one in which the N----H bond of the Schiff base points toward the same membrane surface as the vector from the Schiff base to the cyclohexene ring is compatible with the known tilt angle of the polyene chain.     

Effect of cross linkers on the bacteriorhodopsin photocycle

A general behavior of bacteriorhodopsin in purple membranes from Halobacterium halobium has been observed upon modification resulting in cross-linking of carboxyl and lysine groups. The rise of the M-intermediate contained two components with approximately 50-50% intensity; its decay showed three components with approximately 25-50-25% intensity respectively in a pH range of 5-9. The significance of these remarkably similar data with respect to the proton translocation mechanism in bacteriorhodopsin is that chemical modification allows us to conclude that disturbing parts of the hypothetical "proton conducting chain" does not inhibit proton translocation.     

Characterization and crystal packing of three-dimensional bacteriorhodopsin crystals

The three-dimensional crystals of the integral membrane protein bacteriorhodopsin have been characterized by X-ray diffraction and freeze-fracture electron microscopy: the needle-like form A crystals belong to space group P 1 (pseudohexagonal) with seven molecules per crystallographic unit cell forming one turn of a non-crystallographic helix. The probable arrangement of the bacteriorhodopsin molecules is derived from freeze-fracture electron micrographs and chromophore orientation. Membrane-like structures are not present. The same helices of bacteriorhodopsin molecules found in crystal form A also make up the cube-like crystal form B. They are now arranged in all three mutually perpendicular directions. These cubes are always highly disordered, since the unit cell length corresponds to 6.7 molecules of the 7-fold helix. Very often, conversion of bacteriorhodopsin from the three-dimensional crystals into filamentous material occurs.