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The first and second derivatives of dielectric spectra have evidenced the existence of two interacting states of purple membrane (PM) that respond differently to the intensity of illuminating light providing, this way, underlying consequences to the heterogeneous behavior of bacteriorhodopsin (bR). It is of particular interest to note that the rotational diffusion coefficient of PM has exhibited non-linearity versus light intensity. The explored non-linearity in electrical properties beers, thereby, on changes in PM size. The non-linear variations in PM bending might initiate, in consequence, variations in the dipole moment (permanent and induced) and dc-conductivity of PM patches. Proposal based on PM bending has been introduced to correlate the light intensity effect to the PM lipid environment. Modulation of the global structure of PM and, in turn, its electrical properties by an external perturbation (e.g., light) could be of interest in biotechnological applications based on optoelectronic properties of bR.

Glutaraldehyde, aluminum ions and glycerol (that inhibit the M intermediate decay in the wild-type bacteriorhodopsin and azide-induced M decay in the D96N mutant by stabilization of the M(closed)) accelerate the N decay in the D96N mutant. The aluminum ions, the most potent activator of the N decay, induce a blue shift of the N difference spectrum by approximately 10 nm. Protonated azide as well as acetate and formate inhibit the N decay in both the D96N mutant and the wild-type protein. It is concluded that the N intermediate represents, in fact, an equilibrium mixture of the two ('open' and 'closed') forms. These two forms, like M(closed) and M(open), come to an equilibrium in the microseconds range. The absorption spectrum of the N(open) is slightly shifted to red in comparison to that of the N(closed). Again, this resembles the M forms. 13-cis-all-trans re-isomerization is assumed to occur in the N(closed) form only. Binding of 1-2 molecules of protonated azide stabilizes the N(open) form. Existence of the 'open' and 'closed' forms of the M and N intermediates provides the appropriate explanation of the cooperative phenomenon as well as some other effects on the bacteriorhodopsin photocycle. Summarizing the available data, we suggest that M(open) is identical to the M(N) form, whereas M1 and M2 are different substates of M(closed).

The trimeric, asymmetric and sequential model for the cooperative interaction of the bacteriorhodopsin molecules in purple membranes [Zs. Tokaji, Biophys. J. 65 (1993) 1130–1134] is being extended in the paper. Analyses of data from absorption kinetic measurements with preexcitation and green background illumination, and photoselection measurements on oriented samples confirm the main features of this cooperative interaction and support the validity of the extended model. This model includes the observed heterogeneity of the non-excited state of the bacteriorhodopsin molecules in purple membranes, and the agreement with the data suggests that molecules in any other state than the bacteriorhodopsin ground state can alter the photocycle of their neighbors. The presented results seem to contradict other models for the cooperation of the bacteriorhodopsin molecules.

Laser flash-induced photovoltage responses of the D85S and D85T mutants as well as of the wild-type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl−-induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl− translocation in the cytoplasmic direction. Cl− translocation efficiency of these purple forms is much lower than that of the proton transport by the wild-type bacteriorhodopsin. The values of the efficiency do not exceed 15, 8 and 3% for the D85T, D85S and wild-type acid purple form, respectively. Cl− induces an additional electrogenic phase in the photovoltage responses of the D85S mutant and the wild-type acid purple form. This phase is supposed to be associated with the reversible Cl− movement in the extracellular direction. It is interesting that this component is absent in the photovoltage response of the D85T mutant which has, like halorhodopsin, a threonine residue at position 85.

Differential scanning calorimetry was used to compare the thermal behavior of native and delipidated purple membrane fragments at pH values corresponding to purple, blue and acid-purple forms. At neutral pH, delipidation results in a 2.5- to 3-times increase in the cooperativity of the denaturational transition, accompanied by a minor increase in its temperature. At pH values below 5 the delipidated membranes exhibit considerably higher thermal stability than the native membranes. The reversible predenaturational transition observed in the native state is not detectable upon delipidation. There is no strict correlation between color changes upon acidification and deionization of either native or delipidated purple membranes and their thermal stability.

We have measured the current generated by light-activated proton release from bacteriorhodopsin into solution as a function of both pH and ionic strength. We find that proton release into solution decreases with increasing pH with an intrinsic p K a of 8.2 ± 0.2. This pH dependence indicates that the deprotonation of a certain group inhibits or abolishes proton release. Under physiological conditions, this group either releases a proton directly into solution or interacts with the site of proton release. The most immediate candidates for this protonatable species are tyrosine-57, tyrosine-185, arginine-82, and water; acting individually or cooperatively. The salt dependence of the apparent p K a of this group also allows us to calculate the surface charge density of about −5 charges per bacteriorhodopsin, compatible with previous estimates.

Ultraviolet-visible spectroscopy has been used to follow the solubilization of the dark-adapted purple membrane of Halobacterium halobium by Triton X-100. Turbidity of purple membrane fragments and absorbance of bacteriorhodopsin variations during continuous addition of detergent give solubilization profiles exhibiting several break points corresponding to different equilibrium stages of the solubilization process. The present method allows the determination of the detergent to protein + lipid ratio in mixed aggregates at the corresponding break points. It was concluded that, when performed systematically, this technique is a very convenient and powerful tool for the quantitative study of biomembrane-to-micelle transition.

The photocycle of the light-adapted purple membrane was studied with a time-resolved fluorometry apparatus: fluorescence of the sample suspension (greater than 660 nm) was pumped with a 633-nm cw laser and the temporal change induced by a 532-nm pulsed laser was measured with a photon-counting-type transient recorder. The formation and the decay of the O640 intermediate were clearly observed in the pH region between 4.0 and 11.4. A photochemical cycle of N560 was apparently driven in alkaline suspension (pH greater than 9.3). An O-like fluorescent intermediate Q appears and decays with time constants of less than 0.1 ms and 1.7 +/- 0.2 ms, respectively.

Double flash experiments were performed in order to gain information about the characteristics of the N intermediates of the photocycle of bacteriorhodopsin. The N intermediates of wild-type bacteriorhodopsin and mutant T46V were excited at different delay times after the first laser flash which induced the photocycle and the electric responses were registered. These electric signals revealed that charge motions occurred in both cases, though charge translocation, i.e. H+ pumping, could not be observed. The delay time dependence of the electric signals is characterized by two distinct processes corresponding to two substates of the N intermediates.

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple–blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5–7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple–blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H+. At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.

The photocycle of the acid-purple form of bacteriorhodopsin (BR) was studied by multiwavelength absorption kinetic measurements. The data were evaluated using a new method based on the differences in the kinetics of the absorption changes at different wavelengths [Tokaji, Zs. (1995) FEBS Lett. 357, 156–160]. The evaluation indicates that, in contrast to the previous suggestion, the photocycle of the acid purple BR contains an intermediate that, in its spectral and kinetic properties, resembles the N intermediate of the alkaline BR photocycle. © 1997 Federation of European Biochemical Societies.

At high ionic strength, the pH dependence of the M intermediate decay in a photocycle of the D96N mutant bacteriorhodopsin shows a complicated behavior which is found to be due to the coexistence of two pathways of the M conversion. The M decay which dominates at pH 5. This pathway is insensitive to glutaraldehyde and some other similar inhibitors (lutetium ions, sucrose and glycerol). On the other hand, it is sensitive to the pK changes of the group X (Glu-204) in the outward proton pathway. Possibly, the M decay through this pathway represents a reverse H+ transport process (the proton uptake from the external surface) and proceeds via the L intermediate.

Glutaraldehyde treatment leads to the inhibition (i) of the M intermediate decay in wild-type bacteriorhodopsin (bR) and (ii) of the azide-facilitated M decay in the D96N mutant bR. LuCl3 is shown to be a more potent inhibitor of both processes. Glycerol and sucrose are also inhibitors. None of these agents change the linearity of the azide concentration dependency of the M decay in the D96N mutant but they do shift this dependency to higher azide concentrations. It is concluded that the two M forms are in equilibrium. These M forms differ in the accessibility of the Schiff base for azide and, probably, also for water molecules. The above-mentioned agents shift the equilibrium toward the less accessible M form. The data obtained are in line with the model of azide action as the penetrating proton donor and can hardly be realized within the framework of the model of Le Coutre et al. [(1995) Proc. Natl. Acad. Sci. USA 92, 4962–4966] which assumes that a bound anionic form of azide catalyzes proton transfer to the Schiff base.

We measured the flash-induced absorption anisotropies of mutant bacteriorhodopsin (bR), D96N, in the purple membrane suspension. The measured anisotropy decay at 410 nm differed from that at 570 nm. These wavelength-dependent anisotropies show that the motion of absorption dipole of non-excited bR is faster than that of M-intermediate. The motion of non-excited bR is considered as the rotational motion of whole protein in the purple membrane. This fact suggests that the photo-excitation induces the conformational change of the protein and/or the inter-protein interaction within the membrane, which prevents the motion of M-intermediate.

The M intermediate decay in the photocycle of D96N mutant bacteriorhodopsin does not depend on the light intensity of the exciting flash. Cooperative phenomena in the photocycle are revealed after addition of azide causing acceleration of the M decay and making it kinetically well separated from the N decay. Increase in the light intensity induces slight deceleration of the M decay and significant acceleration of the N decay. The data obtained directly confirm our recent model [ Komrakov and Kaulen (1995) Biophys. Chem. 56, 113–119], according to which appearance of the Mslow intermediate in the photocycle of the wild type bR at high light intensity is due to destabilization of the N intermediate leading to the acceleration of the N→M and N→bR reactions.

An unequivocal answer is given to the question of why the reported fluorescence spectra of bacteriorhodopsin (bR568) have been different from one another. The inconsistency is shown to arise from the accumulation of the fluorescent intermediates O and Q (KN) by cw excitation light. Their fractions in the photo-stationary states depend on the excitation power and the suspension pH. We report the intermediate-free fluorescence spectrum of bR568 obtained with a weak excitation source (632.8 nm, 5.3 × 1015 − 1.9 × 1016 photons cm−2 · s−1) and a near-IR sensitive intensified photodiode array system. The fluorescence maxima of the spectra, F(λ) and f(ṽ), are located at 755 ± 10 nm and 12700 ± 200 cm−1, respectively. The spectrum of O is identical to that of the deionized purple membrane bR605(Fmax = 750 ± 5 nm, fmax = 13,000 ± 100 cm−1). Q (KN) exhibits a blue-shifted spectrum more than that of bR568, (Fmax < 720 nm, fmax > 13,400 cm−1).

Bacteriorhodopsin (bR) in the native purple membrane, in wild type expressed in E. coli and reconstituted in lipid vesicles, and its constituted mutants with substitutions of Tyr-185 by Phe all are found to have different visible retinal CD spectra. The results strongly suggest that the environment of the retinal in bR determines the sign and heterogeneity of its visible retinal CD spectrum. This supports the recent proposal that the observed biphasic CD spectrum of bR is due to the superposition of the CD spectra having opposite signs of more than one type of bR rather than due to exciton coupling.

The binding of the isomers (all- trans , 13- cis , 11- cis and 9- cis ) of 5-demethylretinal to bacterioopsin and the light—dark adaptation as well as the light-driven proton pump action of the resulting bacteriorhodopsin analogue were studied. The (5-demethyl)-bacteriorhodopsinis formed ≈ 3-times faster than unmodified bacteriorhodopsin and shows an efficient light-driven proton pump action. These findings show that upon binding of retinal to bacterioopsin the protein forces the chromophore to adopt a more planar ring-chain conformation than in free retinal.

Cation removal or acidification induces a transition of bacteriorhodopsin (BR568) to a blue species (BR605). Fourier transform infrared spectroscopy now reveals that the protein conformational changes accompanying this transition resemble those which occur during the BR568 to M412 transition and associated light-driven proton transport. This and other results suggest that BR605 and M412 both have similar voltage-dependent protein conformations in which a secondary structure is stabilized by a change in the local electric potential sensed by internal charged groups involved in proton transport.

A resonance Raman molecular probe was used to measure the surface potential of membrane fragments which contain bacteriorhodopsin. It is shown that the surface potential of the native membranes is identical to that of bleached bacteriorhodopsin. It is therefore concluded that the secondary interactions between the retinal chromophore and the protein, which are known to exist, do not have a long-range effect on the exposure of the bacteriorhodopsin at the membrane's surface.

The lifetime of the M-intermediate in the bacteriorhodopsin photocycle was remarkably prolonged on drying a suspension of purple membrane, in arginine solution at alkaline pH. The M-intermediate could be completely accumulated under illumination (530 nm) at room temperature. The crystalline structure of purple membranes was retained after this treatment. The lifetime of the M-intermediate was found to be longer than 100 s and depended on the pH of the purple membrane suspension before drying. It was suggested that an interaction between the guanidium group of arginine and an amino acid residue played an important role in the prolongation.

Regeneration of bacteriorhodopsin (bR) from the M intermediate via a new intermediate P has been studied. In the purple sheets treated with 0.015% Triton X-100 (the P → bR transition is suppressed), the α-maximum of absorption of P is located at 560 ± 5 nm, the extinction coefficient being equal to 0.7 ± 0.1 of that of bR. Besides, there is a small but measurable absorbance increase at 330–350 nm, which seems to be due to a β-maximum of 13-cis-retinal residue in P. The similarity of the α-maximum of P and bR suggests the Schiff base nitrogen to be protonated at the M → P transition. The kinetics of P → bR transition measured at the 335 nm absorbance decrease coincides with that of proton uptake accompanying bR regeneration after flash. A scheme is proposed assuming that H+ absorption from the water phase is coupled to the 13-cis → all-trans isomerization of retinal residue in which the Schiff base is already protonated by a proton-donor group of the protein.

A recent report of electrical measurements on oriented bacteriorhodopsin in gels [(1986) FEBS Lett. 195, 164–168] concluded that low concentrations of diamines reversed the direction of the proton pump. Calculations are presented which show that in low diamine concentrations, charge displacements of the counterion atmosphere in the direction opposite to proton pumping are expected following H + ejection. It is also shown that the effect will be sharply reduced by raising the diamine concentration or by adding excess salt, as was observed. Hence it is not necessary to conclude that diamines reverse the direction of the proton pump itself.

The application of a successive annealing procedure to the scanning calorimetric endotherm of the purple membrane from Halobacterium halobium in phosphate buffer, pH 7.5, leads to five thermal transitions beneath the overall endotherm. Circular dichroism and fluorescence experiments have also been carried out with the native membrane heated at the same scan rate as in calorimetric runs (1 °C min ) as well as with previously heated membrane samples. These results, together with others from the literature, have been used to suggest a preliminary explanation of the five thermal transitions.

We developed a new assay system for the measurement of capacitive electric currents generated by ion pumps using the thin polymer film `Lumirror' (Toray Co., Japan). This system enables us to examine the electrogenicity of ion pumps over a wide range of experimental conditions with high reproducibility due to the mechanical and chemical stability, the high electric resistance and the high electric capacitance of the thin polymer film. Using this method, we examined the photoelectric response of wild type bacteriorhodopsin and its D96N mutant over a wide pH range (2.8–10.0). The results were explained in terms of the affinities of the proton binding sites for translocated protons. A possibility that the direction of the proton transfer from the Schiff base was influenced by the protonation/deprotonation state of the surrounding proton binding sites was suggested. We also found that this film can be used as a substrate for atomic force microscopy (AFM) samples and hence the active purple membrane was observed with AFM.

In native purple membrane (PM), there are approximately 1 squalene, 2 glycolipid sulfate (GLS), and 6 phospholipid (PL) molecules per bacteriorhodopsin (BR) monomer [10]. Brief (∼ 2 min) exposure to 0.1% Triton X-100 removes about 25%, 20%, and 6% of squalenes, GLS, and PL, respectively (this paper) while causing profound changes in the BR photocycle, including the loss of ‘photocooperativity’ [1]. The BR photocycle in Triton-treated PM can be restored to near normal behavior by reconstitution with native PM lipids. Isolated squalenes are not effective whereas PL alone partially restores normal photocycle characteristics.

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.

A monoclonal antibody technique for determining the surface morphology of the purple membrane (PM) has been developed. Two types of antibodies for distinguishing the extracellular surface and the cytoplasmic surface of PM were produced by immunizing the corresponding synthetic peptides to mice. The Langmuir-Blodgett (LB) method was employed to form a single-layer two-dimensional array of PM fragments on an electron microscope grid. The LB film was then treated with the antibodies which had been labeled with colloidal gold particles. Electron microscope observations visualized the morphology of the two surfaces of PM to be rough and smooth regions, respectively; thus the orientation of PM fragments was determined.

The reaction of tetranitromethane with bacteriorhodopsin under irradiation was separated into two stages by selecting wavelengths of the irradiation light. The first stage of the reaction was characterized by a blue shift of the absorption spectrum and the second stage was characterized by decrease of the absorption of the visible region. The blue shift at the first stage was not caused by nitration of tyrosine residues but a modification of the chromophore. This conclusion was confirmed by reconstitution experiments and chromophore analysis. Nitration of tyrosine residues progressed at the second stage of the reaction.

A new two step photobleaching process is observed under continuous illumination of bacteriorhodopsin. This photobleaching is considerable even at physiological temperatures and becomes large at 50–60°C. The photobleaching also increases with increasing pH from 7 to 10. We suggest that the bleaching at its final stage could be due to the dissociation of the retinal and a local thermal denaturation-like process. These facts may question the generally held belief that BR is a stable protein in vivo for a long period of time. Our results may have relevance also to practical applications of bacteriorhodopsin where the stability of bacteriorhodopsin is a key issue. In certain instances, the use of bacteriorhodopsin may require cooled conditions. Here, we defined the conditions under which bacteriorhodopsin is stable. The permanent photobleaching offers a new way of picture imaging and information input for bacteriorhodopsin-based optical devices.

The surface potential of the purple membrane was measured by a novel method by using an artificial bacteriorhodopsin whose chromophore was 13-CF3 retinal instead of retinal. When attached to the apoprotein by a Schiff base, the intrinsic pK of the 13-CF3 chromophore is around 7.3. The apparent pK of this pigment depends on the surface potential and thus on the electrolyte concentration. This allowed us to determine the surface charge density using the Gouy-Chapman equation. The surface charge density was found to be −1.65 ± 0.15 × 10−3 electronic charges per A2 or about 2 negative charges/bacteriorhodopsin. This large value for the surface potential probably explains both part of the strong apparent association of divalent cations with the membrane and the effect of low salt concentrations on light-induced proton release from the purple membrane.

A comprehensive understanding of structure-function relationships of proteins requires their structures to be elucidated to high resolution. With most membrane proteins this has not been accomplished so far, mainly because of their notoriously poor crystallizability. Here we present a completely detergent-free procedure for the incorporation of a native purple membrane into a monoolein-based lipidic cubic phase, and subsequent crystallization of three-dimensional bacteriorhodopsin crystals therein. These crystals exhibit comparable X-ray diffraction quality and mosaicity, and identical crystal habit and space group to those of bacteriorhodopsin crystals that are grown from detergent-solubilized protein in cubic phase.

The mechanism of the intramolecular proton transfer in the membrane protein bacteriorhodopsin (bR) is studied. The kinetic isotope effects after H/D exchange were determined for the individual photocycle reactions and used as an indicator. Significant differences in the kinetic isotope effects are observed between the intramolecular proton transfer on the release and the uptake pathways. The results suggest a fast intramolecular proton transfer mechanism in the proton release pathway, which is similar to the one proposed for ice, where the rate limiting step is the proton movement within the H bond. However, the reactions in the intramolecular proton uptake pathway occur in a mechanism similar to the one suggested for liquid water, where the rate limiting step is given by a rotational rearrangement of H bonded network groups. We propose that the experimental evidence for a proton wire mechanism given here for bacteriorhodopsin is of general relevance also for other proton transporting proteins.

The mapping of flexible protein domains with the atomic force microscope is reviewed. Examples discussed are the bacteriorhodopsin from Halobacterium salinarum, the head-tail-connector from phage φ29, and the hexagonally packed intermediate layer from Deinococcus radiodurans which all were recorded in physiological buffer solution. All three proteins undergo reversible structural changes that are reflected in standard deviation maps calculated from aligned topographs of individual protein complexes. Depending on the lateral resolution (up to 0.8 nm) flexible surface regions can ultimately be correlated with individual polypeptide loops. In addition, multivariate statistical classification revealed the major conformations of the protein surface.

Glutaraldehyde, lutetium ions and glycerol inhibit the blue shift of the difference spectra maximum of the M intermediate in the D96N mutant. The M formed has a spectrum indistinguishable from the M intermediate in wild-type bacteriorhodopsin. It has been concluded that the M open form previously described by us is identical to the M2 and M n intermediates postulated by Zimanyi et al. ( Photochem. Photobiol. (1992) 56, 1049–1055) and Sasaki et al. ( J. Biol. Chem. (1992) 267, 20782–20786), respectively. It is supposed that its formation is accompanied by the appearance of the cytoplasmic proton half-channel. M open in the wild-type protein is present in a very low amount due to the shift of the M closed ↔M open equilibrium towards the M closed . The inhibitors used do not prevent the multiphase pattern of the M formation in either mutant or wild-type proteins.

The photocycle and the proton pumping kinetics of bacteriorhodopsin, as well as the transfer rate of protons from the membrane surface into the aqueous bulk phase were examined for purple membranes in water and in ice. In water, the optical pH indicator pyranine residing in the aqueous bulk phase monitors the H+-release later than the pH indicator fluorescein covalently linked to the extracellular surface of BR. In the frozen state, however, pyranine responds to the ejected H+ as fast as fluorescein attached to BR, demonstrating that the surface/bulk transfer is in ice no longer rate limiting. The pumped H+ appears at the extracellular surface during the transition of the photocycle intermediate L550 to the intermediate M412. The Arrhenius plot of the M formation rate suggests that the proton is translocated through the protein via an ice-like structure.

Light-scattering changes were found to accompany the bacteriorhodopsin photocycle in a suspension of purple membranes. A light-scattering signal comprises three phases, connected with (i) bR → M transition, (ii) transition of M to P intermediate (other names: N,R350 intermediate), (iii) P → bR transition. Light-scattering changes seem to result from changes in the purple membrane shape in response to the bacteriorhodopsin conformational changes during the photocycle.

The structure of the M-intermediate appearing in the photo-cycle of bacteriorhodopsin was studied with X-ray diffraction techniques at room temperature. The lifetime of the M-intermediate was prolonged by treatment with an arginine solution at alkaline pH (Nakasako et al.,FEBS Lett. 254, 211–214). The diffraction profile of membranes which had accumulated the M-intermediate had small but significant differences in the intensities of Bragg reflections and the lattice constant in comparison with that of membranes havingtrans-bacteriorhodopsin. Diffraction intensities were carefully evaluated and the structural changes during the formation of the intermediate were evaluated with difference Fourier analysis. We could find structural changes around helices G and B.

A suspension of freeze-thawed mitochondria mixed with purple membranes from Halobacteria was illuminated with visible light. It was found that the light exposure prevented inhibition of succinate oxidation. The illumination also led to a decrease in inhibition of the rate of ferricyanide reduction by rat liver mitochondria in the presence of succinate. Both phenomena are explained by the fact that oxalacetate inhibition of succinate dehydrogenase is prevented by light-induced energization of mitochondrial membranes due to the contact with purple membranes.

The orientation of purple membrane in gels for photoelectric measurements is relatively poor, when they are prepared with the standard technique of applying a DC electric field and rapid polymerization. We have improved it by adding a high magnetic field (17.5 T) and increasing the viscosity of the membrane suspension. This process has resulted so far in a 3-fold increase of the photoelectric signals obtained. The magnetic susceptibility of purple membrane was determined.