A study of light-induced conductivity in bacteriorhodopsin

Measurements have been made of light-induced conductivity changes and the associated kinetics of the relaxation processes in aqueous suspensions and sonicated liposomes containing bacteriorhodopsin (bR). Aqueous suspensions exhibit a single relaxation time of 1 to 2 ms. The addition of D₂O to the aqueous suspension slows down the relaxation time, fourfold. Similar behaviour is seen in sonicated liposomes with a relaxation time of 2 to 3 ms. Activation energies of approximately 14 and 6 kJM^-1 are obtained for the effect in sonicated liposomes and aqueous suspension containing bR, respectively. These relaxation processes with lifetime of 1 to 2 ms suggest conformational changes in the protein moiety of bR which most probably may be associated with protonation-deprotonation processes or less likely the release and binding of small ions.     

Kinetic study into the irreversible thermal denaturation of bacteriorhodopsin

We report on a differential scanning calorimetry study of native purple membranes under the following solvent conditions: 50 mM carbonate-bicarbonate, 100 mM NaCl, pH 9.5 and 190 mM phosphate, pH 7.5. The calorimetric transitions for bacteriorhodopsin denaturation are highly scanning-rate dependent, which indicates that the thermal denaturation is under kinetic control. This result is confirmed by a spectrophotometric study on the kinetics of the thermal denaturation of this protein. The calorimetric data at pH 9.5 conform to the two-state irreversible model. Comments are made regarding the information obtainable from differential scanning calorimetry studies on bacteriorhodopsin denaturation and the effect of irreversibility on the stability of membrane proteins.Correspondence to: J. M. Sanchez-Ruiz     

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.     

A study on the fabrication of chemically modified bacteriorhodopsin film and its photochemical properties

Chemically modified bacteriorhodopsin (BR) films embedded in gelatin matrix were fabricated. It was found that chemically modified BR/gelatin film remained the original conformation of BR and possessed homogeneity. Then, the photochemical conversion from all-trans O state to 9-cis P state in the chemically modified BR film/gelatin was investigated. For comparison purpose, the same conversion in the wild-type BR (BRwt) film was also presented here. To our know, it is the first report to show that the absorption changes of the P state in the chemically modified BR film were larger than in the BRwt film upon illumination with red light at the low actinic power density.     

Fast electric response signals in the bacteriorhodopsin photocycle

The time behavior of flash-induced charge movements during the first steps in the bacteriorhodopsin photocycle was measured on a suspension of purple membranes oriented by an electric field. The experiments were done in the temperature range 80–278 K. During the formation of the intermediate K, two negative (with respect to the direction of the proton pump) components of the response signal are well resolved with time constants τ₁ < 3 μs and $\text{τ}{}_2\text{? 150 μ}\text{s}$ at 200 K. The distances of the charge displacements responsible for the electric signals are estimated. On the basis of the results the two components are assigned to two steps in the trans-cis isomerization of the retinal. A third negative component appears at higher temperatures which is related by time constant measurements to the K → L transition.     

Electrooptic study of the deionized form of bacteriorhodopsin

Electric field induced light scattering by suspensions of cation-depleted purple membranes, obtained by deionization of purple membrane (PM) suspensions on a cation exchange column or by electrodialysis at a pH around 6, shows a strong drop (more than 5 times) in the value of the permanent dipole moment relative to that of PM fragments. The membrane dipole moments were measured both at low dc and ac electric fields as well as by using electric field pulses with reversing polarity. Some slight changes in the dispersion of the electric polarizability were also observed.Microelectrophoretic measurements showed that the electric charge of the membrane fragments is increased by 30% after deionization. The importance of these data for the understanding of the blue membrane properties and subsequently for the mechanism of proton pumping are discussed.     

Proton transfers in the bacteriorhodopsin photocycle

The steps in the mechanism of proton transport in bacteriorhodopsin include examples for most kinds of proton transfer reactions that might occur in a transmembrane pump: proton transfer via a bridging water molecule, coupled protonation/deprotonation of two buried groups separated by a considerable distance, long-range proton migration over a hydrogen-bonded aqueous chain, and capture as well as release of protons at the membrane-water interface. The conceptual and technical advantages of this system have allowed close examination of many of these model reactions, some at an atomic level.     

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.     

Retinal-protein interactions in bacteriorhodopsin monomers,dispersed in the detergent L-1690

Bacteriorhodopsin monomer dispersed in a solution of the detergent L-1690 could maintain the specific interaction between retinal and protein in the pH range 9.0-0.0 at 25°C. λ_max of the absorbance spectrum was 550 nm at pH 9.0, 556 nm at pH 5.5, 609 nm at pH 2.1 and 570 nm at pH 0.0. Increasing the NaCl concentration in the solution promoted formation of the 609 nm product at pH 5.0-3.0 and also its transition to the 570 nm product at pH 2.5-1.0. Retinal isomer analysis gave a ratio of 13-cis- to all-trans-retinal of 53 : 47 at pH 5.5. When the pH of the solution was reduced, the relative content of all-trans-retinal increased and the ratio of 13-cis- to all-trans-retinal was 14 : 86 at pH 0.0. Illumination of the solution at pH 7.2 yielded a product containing 9-cis-retinal or 9-cis, 13-cis-retinal, which may be due to a reaction other than the photoreaction cycle.     

Carbodiimides inhibit the acid-induced purple-to-blue transition of bacteriorhodopsin

Reaction of purple membrane with water soluble carbodiimides inhibits the spectral transition from purple to blue observed at acid pH. The pK and Hill constant for this transition are shifted from 3.4 to 2.6 and from 1.8 to 0.85, respectively. The results suggest a connection between the uptake side of the proton pump and the purple-to-blue transition.     

Time-resolved refractive index change during the bacteriorhodopsin photocycle

Kinetic refractive index spectroscopy has been applied to the study of the bacteriorhodopsin photocycle. A fully hydrated purple membrane film was examined in the temperature range from 10° to 40°C using 532 nm excitation (doubled Nd YAG laser) and 633 nm (He–Ne laser) testing beam. Multiexponential fitting of the data revealed five processes. Four of them are well known from kinetic optical absorption studies. The fifth process has only recently been observed in optical absorption experiments where it has a relatively small amplitude. In our refractive index experiments it has an amplitude of up to 30% of the full signal amplitude. It is characterized by an Arrhenius temperature dependence with an activation enthalpy of 40±5 kJ/mol and a decay time of about 0.8 ms at 20°C.     

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.     

Optical picosecond studies of bacteriorhodopsin containing a sterically fixed retinal

The photochemical behaviour of an analogous bacteriorhodopsin (9,12-Ph-BR) which contains the sterically fixed 9,12-phenylretinal has been investigated with picosecond spectroscopy. The following results have been obtained. No ground-state intermediate photoproduct is found in agreement with the previous observation that 9,12-Ph-BR does not exhibit proton pumping under illumination. The excited singlet state has a lifetime of τ_S = 10 ± 2 ps. This lifetime agrees favourably with the value calculated from the radiative lifetime τ_rad = 6.2 ns and the fluorescence quantum efficiency of 1.2·10⁻³. Excited-state absorption occurs which results in fluorescence in the ultraviolet region. These various observations differ drastically from the corresponding findings on bacteriorhodopsin. Most important for an understanding of the differences is the fact that 9,12-phenylretinal does not isomerize in the protein's binding site in contrast to retinal. Our data therefore suggest that the formation of the intermediate K observed in bacteriorhodopsin is accompanied by the all-trans to 13-cis isomerization.     

Effects of electric field on the photocycle of bacteriorhodopsin

The photoconversion of bacteriorhodopsin and the effects of an applied electric field (5 · 10⁷ V · m^?1) were studied in dry films of purple membranes from Halobacterium halobium. The electric field was found to cause at least two different effects: (1) it blocks in part the formation of the batho-bacteriorhodopsin (K), most probably due to electrically-induced dark transition of some bacteriorhodopsin molecules into the photochemically inactive form; (2) it decreases the rate of the intermediate M decay, the rise time of the M formation being unaffected by electric field. The observed phenomena may suggest a feedback control mechanism for the regulation of the bacteriorhodopsin photocycle in purple membranes.     

Electrooptical analysis of blue and of cation-regenerated bacteriorhodopsin

Blue bacteriorhodopsin was prepared by electrodialysis, cation-exchange chromatography and acidification. The electrooptical properties of these preparations compared to those of the native purple bacteriorhodopsin suggest that the blue bacteriorhodopsin has a smaller induced dipole moment than the native purple bacteriorhodopsin and that bound cations in the native bacteriorhodopsin stabilize the protein conformation in the membrane.Purple bacteriorhodopsin was regenerated by addition of potassium, magnesium or ferric ions to blue bacteriorhodopsin. Both spectrscopically and electrooptically the potassium- and ferric-regenerated samples are different from the native purple state. Although the magnesium-regenerated sample is spectroscopically similar to the native purple bacteriorhodopsin, the electrooptical properties are rather similar to those of the cation-depleted blue sample, suggesting that it is very difficult to re-stabilize protein structures once cations are depleted.     

Protein rotation and chromophore orientation in reconstituted bacteriorhodopsin vesicles

Bacteriorhodopsin has been reconstituted into lipid vesicles with dipalmitoyl and dimyristoyls phosphatidylcholine. Circular dichroism (CD) measurements show that the proteins are in a monomeric state above the main lipid phase transition temperature (T_c), 41 and 23°C for dipalmitoyl and dimyristoyl phosphatidylcholine, respectively. Below T_c, the CD spectrum is the same as that found for the purple membrane. The latter result implies that the orientation of the chromophore at these temperatures is most likely the same as in the purple membrane ($\text{70° ± 5°}$ from the normal to the membrane plane).Transient dichroism measurements show that below T_c the proteins are immobile, while above this temperature protein rotation around an axis normal to the plane of the membrane is occurring. In addition, from the data the angle of the chromophore for the rotating proteins with respect to the rotational diffusion axis can be calculated. This angle is found to be $\text{30° ± 3°}$ and $\text{29° ± 4°}$ in dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, respectively. This is considerably smaller than the value of $\text{70° ± 5°}$ for the natural biomembrane. A reversible reorientation of the chromophore above and below the respective main T_c transition temperature could explain the change of angle observed provided that all the molecules rotate above T_c.     

Electrooptical studies on proton-binding and -release of bacteriorhodopsin

Electric field induced pH changes of purple membrane suspensions were investigated in the pH range from 4.1 to 7.6 by measuring the absorbance change of pH indicators. In connection with the photocycle and proton pump ability, three different states of bacteriorhodopsin were used: (1) the native purple bacteriorhodopsin (magnesium and calcium ions are bound, the M intermediate exists in the photocycle and protons are pumped), (2) the cation-depleted blue bacteriorhodopsin (no M intermediate), and (3) the regenerated purple bacteriorhodopsin which is produced either by raising the pH or by adding magnesium ions (the M intermediate exists). In the native purple bacteriorhodopsin there are, at least, two types of proton binding sites: one releases protons and the other takes up protons in the presence of the electric field. On the other hand, blue bacteriorhodopsin and the regenerated purple bacteriorhodopsin (pH increase) show neither proton release nor proton uptake. When magnesium ions are added to the suspensions; the field-induced pH change is observed again. Thus, the stability of proton binding depends strongly on the state of bacteriorhodopsin and differences in proton binding are likely to be related to differences in proton pump activity. Furthermore, it is suggested that the appearance of the M intermediate and proton pumping are not necessarily related.     

The role of proline residues in the dynamics of transmembrane helices: the case of bacteriorhodopsin

Proline residues in transmembrane helices have been found to have important roles in the functioning of membrane proteins. Moreover, Pro residues occur with high frequency in transmembrane α-helices, as compared to α-helices for soluble proteins. Here, we report several properties of the bacteriorhodopsin mutants P50A (helix B), P91A (helix C) and P186A (helix F). Compared to wild type, strongly perturbed behaviour has been found for these mutants. In the resting state, increased hydroxylamine accessibility and altered Asp-85 pK_a and light-dark adaptation were observed. On light activation, hydroxylamine accessibility was increased and proton transport activity, M formation kinetics and FTIR difference spectra of M and N intermediates showed clear distortions. On the basis of these alterations and the near identity of the crystalline structures of mutants with that of wild type, we conclude that the transmembrane proline residues of bacteriorhodopsin fulfil a dynamic role in both the resting and the light-activated states. Our results are consistent with the notion that mutation of Pro to Ala allows the helix to increase its flexibility towards the direction originally hindered by the steric clash between the ring Cγ and the carbonyl O of the i-4 residue, at the same time decreasing the mobility towards the opposite direction. Due to their properties, transmembrane Pro residues may serve as transmission elements of conformational changes during the transport process. We propose that these concepts can be extended to other transmembrane proteins.     

Towards structural investigations on isotope labelled native bacteriorhodopsin in detergent micelles by solution-state NMR spectroscopy

¹H NMR signals of the retinal moiety in detergent-solubilizedbacteriorhodopsin are assigned, enabling the interpretation of NOEs within thechromophore. To achieve this, a number of differently labelled samples wereprepared to test the applicability of the various assignment and distancemeasurement strategies. In measurements with and without light,¹H and ¹³C chemical shifts of the retinal in thenative protein were partially assigned for both the dark- and thelight-adapted states. Additionally, samples with residue-specific¹H amino acids and/or retinal in an otherwise deuterated proteinwere prepared to measure the distances between either two kinds of amino acidsor between individual amino acids and the retinal moiety. With the observationof NOE within the bound retinal and between retinal and its neighbouring aminoacids, an important step towards the elucidation of distance constraints inthe binding pocket of the proton pump is made.     

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.