Purple Membrane Suspensions Research Articles

Chloride- and pH-dependent proton transport by BR mutant D85N.

Photocurrents from purple membrane suspensions of D85N BR mutant adsorbed to planar lipid membranes (BLM) were recorded under yellow (lambda > 515 nm), blue (360 nm < lambda < 420 nm) and white (lambda > 360 nm) light. The pH dependence of the transient and stationary currents was studied in the range from 4.5 to 10.5. The outwardly directed stationary currents in yellow and blue light indicate the presence of a proton pumping activity, dependent on the pH of the sample, in the same direction as in the wild-type. The inwardly directed currents in white light, due to an inverse proton translocation, in a two-photon process, show a pH dependence as well. The stationary currents in blue and white light are drastically increased in the presence of azide, but not in yellow light. The concentration dependence of the currents on azide indicates binding of azide to the protein. In the presence of 1 M sodium chloride, the stationary proton currents in yellow light show an increase by a factor of 25 at pH 5.5. On addition of 50 mM azide, the stationary current in yellow light decreases again, possibly by competition between azide and chloride for a common binding site. The observed transport modes are discussed in the framework of the recently published IST model for ion translocation by retinal proteins [U. Haupts et al., Biochemistry 36 (1997) 2-7].

Chromophore reorientations in the early photolysis intermediates of bacteriorhodopsin

The photoselection-induced time-resolved linear dichroism of a bacteriorhodopsin suspension of purple membrane from 350 to 750 nm is measured by a new pseudo-null measurement technique. In combination with time-resolved absorption measurements, these linear dichroism measurements are used to determine the reorientation of the retinal chromophore of bacteriorhodopsin from 50 ns to 50 microseconds after photolysis. This time range covers the times when the K photointermediate decays to form L, as well as the early times during the formation of the M intermediate in the photocycle. An analysis of the photoselection-induced linear dichroism measured directly, along with the absorbance changes polarized parallel to the linearly polarized excitation, shows that the anisotropy is invariant over this time period, implying that the photolyzed chromophore rotates less than 8 degrees C with respect to unphotolyzed chromophores during this part of the photocycle.

Evidence of Octopolar Symmetry in Bacteriorhodopsin Trimers by Hyper-Rayleigh Scattering from Purple Membrane Suspensions

The hyper-Rayleigh scattering technique has been applied to suspensions of purple membranes, and the size distribution of these membranes, deposited on a mica surface, has been determined by means of atomic force microscopy. A model is developed to express the first hyperpolarizability of a purple membrane fragment in terms of the first hyperpolarizability of the protein bacteriorhodopsin and the angle of the retinal protonated Schiff base with the surface of the membrane. By measuring the depolarization ratio of the scattered second-harmonic light, we deduce a value of (10° ± 1°) for the magnitude of this angle. The arrangement of the proteins in the membrane is found to be predominantly octopolar. Using this angle and the average size of the purple membrane fragments, we find a value of (2000 ± 400) × 10-30 esu for the hyperpolarizability of the protein if we assume that the proteins in a purple membrane patch can be treated as correlated scatterers. Alternatively, using a value of 2100 × 10-30 esu for the hyperpolarizability of the protein, we calculate the average number of proteins in a purple membrane patch (2530). This average is in good agreement with the median (2270) and the average (3470) of the size distribution of the membranes.

Culture temperature affects the molecular motion of bacteriorhodopsin within the purple membrane.

We measured the absorption anisotropies of bacteriorhodopsin (bR) within a purple membrane suspension after photo-excitation in the millisecond time range. The purple membranes used were isolated from Halobacterium salinarium grown at three different culture temperatures, 37.0, 43.0 and 47.5 degrees C. For the membranes from the 37.0 degrees C culture, the observed anisotropies at wavelengths of 410, 570 and 680 nm showed almost the same slow decay. The slow decaying of the anisotropies originated from the rotation of the membrane itself. Using the membranes from the 43.0 and 47.5 degrees C culture, however, we found that the anisotropy change varied at each wavelength measured. In these cases, it is shown from detailed data analysis that 1) the rotational motion of photo-intermediates within the membrane is more restricted than that of non-excited bR and 2) the distorted arrangements of the proteins within the membrane remain, even after photo-intermediates return to ground-state bR. This restricted motion is probably caused by the conformational changes in photo-intermediates, which prevent the rotation of the monomer protein and/or lead photo-intermediates to bind with neighboring proteins.

The molecular motion of bacteriorhodopsin mutant D96N in the purple membrane

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.

Temperature dependence and kinetic aspects of proton release and uptake by purple membrane

Transient electric conductivity changes were detected on electric field aligned purple membrane suspension. The electric signal arises from the proton release and uptake by the purple membrane during the photocyle of bacteriorhodopsin. This electric signal contains several components. Two components can be distinguished with a short and a longer time delay similar to the fast and slow components of the M state. Both components contain proton release and uptake and were kinetically analysed; the temperature dependences of the rate constants were determined. In our interpretation the rate constants are related to the lifetimes of surface proton binding sites for release and uptake and the time delays to the inner proton movement inside the membrane. Two possible interpretations of the two components are discussed, one in connection with a sequential photocycle with back reactions, the other assuming two primary proton acceptors for the Schiff base.

Theory of second harmonic generation in randomly oriented species

Abstract It is well known that second harmonic generation (SHG) is a process forbidden within atomic and molecular fluids. Nonetheless recent experimental observations of second harmonic evolution in suspensions of randomly oriented Halobacterium halobium purple membranes have raised new questions about the precise criteria which determine prohibition of the second harmonic. To address the problem a theoretical framework for SHG is developed that specifically deals with molecular systems, and is therefore cast in terms of molecular properties with more regard to the influence of the local structure. This contrasts with the classical approach based on bulk susceptibilities, which has not proved adequate to explain the conflicting experimental results. By properly formulating the detailed procedure for dealing with the necessary orientational averages, the present theory discloses a relationship between the coherent process of second harmonic generation and a directed component of its incoherent counterpart, hyper-Rayleigh scattering. Inter alia, the theory explains the SHG detected in purple membrane suspensions. The polarisation features of the harmonic evolution are also considered more generally, and in particular it is shown that the SHG signal will persist under conditions of circularly polarised pumping. This specific polarisation feature will allow experimental validation of the theory.

Reorientations in the bacteriorhodopsin photocycle.

Reversible photoinduced reorientations of bacteriorhodopsin have been detected in suspensions of the purple membrane of Halobacterium salinarium. The anisotropy in bacteriorhodopsin during the nanosecond through millisecond stages of the photocycle was measured by time-resolved linear dichroism and transient absorption measurements. From these measurements the anisotropies of the K, L, M, and O intermediates were determined and related to the chromophore orientation with respect to the initially selected orientation. The anisotropies of the K and L states are 0.38 +/- 0.01 and 0.35 +/- 0.01, respectively. Further anisotropy decay after formation of the M intermediate in about 0.5 ms is evidence of orientational motion at this stage in the photocycle. A constant anisotropy with a value of 0.39 +/- 0.02 in the O intermediate demonstrates a recovery of the initial protein orientation with the formation of the O state. These results demonstrate that reorientations in BR are photoinduced and reversible. Similar measurements for L and M were carried out for purple membrane in polyacrylamide gels, where the anisotropies in the L and M states are 0.38 +/- 0.014 and 0.36 +/- 0.01, respectively. These results show that reorientations also occur in BR immobilized in gels. Anisotropy decay in the M state after formation of the M intermediate was not detected in the gels, in contrast to the M intermediate in suspensions. Orientational changes are observed for BR in purple membrane suspensions in the K state, during the K-->L step, in the M state possibly related to an M1-->M2 transition, and in the O state, where an almost complete return to the original orientation occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibody-Mediated Bacteriorhodopsin Orientation for Molecular Device Architectures

A rational method for constructing highly oriented films of purple membrane (PM) has been established by using two kinds of bispecific antibodies with different antigen-binding sites, one binding to a specific side of bacteriorhodopsin and the other to a phospholipid hapten. A hapten monolayer deposited on a metal electrode was treated with a bispecific antibody solution and incubated with a PM suspension to produce a highly oriented PM film, as confirmed by electron microscopy in which an immunogold labeling technique was used. This antibody-mediated PM monolayer was then used in the construction of a light-sensing photoelectric device. A comparison of the two incorporated PM monolayers showed that highly efficient photocurrents were produced by the PM monolayer whose unidirectionally oriented cytoplasmic surface faces the electrode.

Hyper-Rayleigh light scattering from an aqueous suspension of purple membrane

Here we report the first observation of hyper-Rayleigh light scattering from bacteriorhodopsin in the form of an aqueous suspension of unoriented purple membranes. A typical purple membrane suspension used in our experiments contains approximately 10(8) randomly oriented purple membranes. Each purple membrane contains approximately 10(5) bacteriorhodopsin molecules in a two-dimensional crystallinearray. Hyper-Rayleigh light scattering is observed when the purple membrane suspension is illuminated with light that has a wavelength of 1064 nm. We propose that the 532-nm scattered light from each of the bacteriorhodopsin molecules in a single purple membrane is coherent, and that the scattered light from different purple membranes is incoherent. This proposal is supported by the following experimental observations: (a) the 532-nm light intensity is proportional to the square of the incident power, (b) the intensity of the 532-nm signal is linearly proportional to the concentration of purple membrane in solution, (c) the scattered 532-nm light is incoherent, (d) the scattered 532-nm light intensity decreases if the size of the purple membranes is reduced while the bacteriorhodopsin concentration is kept constant, and (e) the 532-nm light is due to the retinal chromophore of the bacteriorhodopsin molecule. The ratio of horizontal polarized hyper-Rayleigh scattered light to vertically polarized hyper-Rayleigh scattered light gives the angle (23 ± 4°) of the retinal axis with respect to the plane of the purple membrane. The hyperpolarizability of the bacteriorhodopsin molecule is found to be 5 ± 0.4 × 10(-27) esu.

EFFECT OF MELITTIN ON PHOTOCYCLE AND PHOTOPOTENTIAL OF PURPLE MEMBRANE: SITES OF INTERACTION BETWEEN BACTERIORHODOPSIN AND MELITTIN

Abstract— Purple membrane (PM) suspension and artificial bilayer lipid membranes (BLM) containing PM sheets were treated with melittin. Both the decaying of the photocycle intermediate M412 and proton translocation were inhibited by melittin: The yields and rate of the slow‐decaying component of M412 (M412s) together with the proton release and its uptake rate were significantly decreased, but the rate of the fast‐decaying component of M412 (M4120 had only slight changes. Relatively high concentrations of melittin could cause aggregation in PM suspensions. Addition of melittin to a BLM solution increased the continuous photopotential signal but decreased the transient signal. We suggest that there might exist strong interactions between melittin and bacteriorhodopsin in addition to the melittin–lipid action. On the other hand, the results also indicate that proton translocation was more likely to be coupled with M412s and both were more sensitive to the changes caused by the melittin–PM interaction than was M412f.

Resonance Raman and optical transient studies on the light-induced proton pump of bacteriorhodopsin reveal parallel photocycles.

The photocycle of bacteriorhodopsin (bR) was studied at ambient temperature in aqueous suspensions of purple membranes using time-resolved resonance Raman (RR) and optical transient spectroscopy (OTS). The samples were photolyzed, and the fractional concentrations of the retinylidene chromophore in its parent state, BR570, and in the intermediate states L550, M412, N560, and O640 were determined in the time domain 20 microseconds-1 s and in the pH range 4-10.5. Two kinetically different L components could be identified. At pH 7 one fraction of L (approximately 65%) decays in 80 microseconds to M (deprotonation of the Schiff base), whereas the residual part is converted in approximately 0.5 ms to N. The RR spectra reveal only minor structural changes of the chromophore in the L-->N transition. These were attributed to a conformational change of the protein backbone [Ormos, P., Chu, K., & Mourant, J. (1992) Biochemistry 31, 6933]. With decreasing pH the L-->N transition is delayed to > 2 ms following a titration-like function with pKa approximately 6.2. The decay of M412 monitored by OTS can be fitted for each pH value by two different amplitudes and time constants (Mf, tau f; Ms, tau s; f = fast, s = slow). Both Mf and Ms consist of subcomponents which can be distinguished by their different reaction pathways (but not by OTS). Mf occurs in the reaction sequences L-->Mf-->N-->BR and L-->Mf-->O-->BR. The population of the first sequence, in which N is formed with the time constant tau f (approximately 2-4 ms, pH 6-10.5), increases with pH. Ms is also found in two different reaction sequences of the form L-->Ms-->BR. The quantitative analysis reveals that each "titration effect" can be related to a certain fraction of bR. It is proposed that each fraction can be identified with a "subspecies" of bR which undergoes an independent and individual cyclic reaction. A complete reaction scheme is set up which represents the manifold of observed phenomena. It is concluded from the pH dependence of the lifetimes of Ms and N that the reconstitution of BR570 in the reaction steps Ms-->BR and N-->BR requires the uptake of a proton from the external phase. It is argued that this proton catalyzes the reisomerization of retinal, whereas the Schiff base is internally reprotonated from Asp-85. A model for proton pumping is proposed in which the proton taken up from the external phase to catalyze the reisomerization of retinal is the one which is pumped through the membrane during the photocycle of bR.

Pyroelectric Current of Oriented Purple Membrane Films

By applying an electric field (2 to 5.5 Volt/mm) to a suspension of purple membrane from Halobacterium halobium, oriented purple-, purple blue-, and blue-colored membrane films have been formed on conductive glasses. Pyroelectric currents have been found for these three types of oriented films at temperatures between 25 and 160°C. The oriented purple-colored membrane (PM) film has positive current peaks at around 60, 80, and 115°C and reverses its sign at around 130°C, followed by a negative peak at 145°C. (Instead of the positive peak at 80°C, a negative peak has been found around 90°C for purple blue- and blue-colored membrane films.) The current peak at 80°C for the PM film could be due to a conformational change in the protein, which includes at least a change in the environment of the chromophore. The sign-reversal current at 130°C for the PM film seems to be related to denaturation of the protein. These peak temperatures in the pyroelectric current of the PM films, i.e., 60, 80, and 115°C, have been found to agree well with some characteristic temperatures observed by Shnyorov et al. (D-H exchange), Jackson et al. (DSC and absorption spectra of visible light), and Hiraki et al. (X-ray, absorption spectra ).

Fourier transform infrared analysis of bacteriorhodopsin secondary structure.

Fourier transform infrared spectroscopy at a resolution of 1 cm-1 has been used to study the conformation of dark-adapted bacteriorhodopsin in the native purple membrane, in H2O and D2O suspensions. A detailed analysis of the amide I bands was made using derivative and deconvolution techniques. Curve-fitting results of four independent experiments indicate, after estimation of the methodological errors, that native bacteriorhodopsin contains 52-73% alpha-helices, 13-19% reverse turns, 11-16% beta-sheets, and 3-7% unordered segments. Our analysis has enabled the identification of several components corresponding to alpha-helices, beta-sheets, and reverse turns. Besides the alpha I- and alpha II-helices (peaking at 1658 and 1665 cm-1), we propose that two more infrared bands arise from alpha-helical structures: one at 1650 cm-1 from alpha I and another one at 1642 cm-1 in H2O suspension, which could originate from type III beta-turns (i.e., one turn of 3(10)-helix). The relatively high content of reverse turns suggests the presence of one reverse turn per loop, plus another one in the C-terminal segment. On the other hand, several reasons argue that the calculated mean beta-sheet content of around 14% should be decreased somewhat. These beta-sheets could be located in the noncytoplasmatic links of the bacteriorhodopsin molecule.

Oriented purple membrane multilayers of halobacteria fabricated by langmuir–blodgett and electrophoretic sedimentation techniques

AbstractOrganised films containing oriented patches of purple membranes and consisting of 15–75 layers and approximately 1000 layers were fabricated by the Langmuir–Blodgett and electrophoretic sedimentation techniques respectively. ‘Surface pressure vs. molecular area’ isotherms for monolayers of purple membranes suspended in hexane and deposited on the air/water interface were studied. Circular dichroism and absorption spectroscopy were used to investigate the effect of an organic solvent on the chromophore binding site in the purple membranes. Kinetic methods based on flash excitation were employed to determine the influence of the film deposition conditions on the photoelectric response of bacteriorhodopsin and on the M412 intermediate relaxation. Structural characteristics for multilayers prepared from water and hexane purple membrane suspensions were determined by low‐angle X‐ray scattering. The influence of the layer deposition conditions on the electrical, physical and photoelectrical properties of ordered structures containing bacteriorhodopsin is discussed.

TIME‐RESOLVED ULTRAVIOLET ABSORPTION CHANGES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN*

Abstract– The kinetics of the absorption changes associated with the perturbation of aromatic acids during the photocycle of bacteriorhodopsin (bR) were studied at room temperature with microsecond time‐resolution. Flash experiments with nanosecond excitation at 532 nm were performed on the purple membrane suspension at a number of measuring wavelengths in the spectral range250–630 nm (to monitor both non‐chromophore changes and the photocycle kinetics). The kinetic data collected at different wavelengths were simultaneously fitted with a sum of exponentials to obtain time‐resolved UV‐VIS difference spectra of photocycle intermediates. This approach allowed us to separate kinetically distinct contributions coupled with tryptophan(s) and tyrosine(s) perturbations. Contributions associated with a reversible perturbation of tryptophans appeared with complex (multistep) kinetics during the bRM transitions and relaxed in a single step during the M0 transition. A contribution associated with perturbation of the local environment of tyrosine appeared before the L and relaxed during the Ob&amp;#x030a; transition.

Distortions in the photocycle of bacteriorhodopsin at moderate dehydration

The photoreaction of bacteriorhodopsin was studied in moderately dehydrated films (relative humidities between 100 and 65%). Time-resolved difference spectra from a gated optical multichannel analyzer, between 100 ns and 100 ms after photoexcitation, were decomposed into sums of difference spectra of the intermediates K, L, M, N, and O, and the kinetics obtained were fitted to various alternative schemes. The data confirm the model of a single reaction sequence with reversible reactions we proposed recently for purple membrane suspensions (Váró, G., and J. K. Lanyi. Biochemistry. 1990. 29:2241-2250) but including reversibility also for the reaction K in equilibrium with L in addition to L in equilibrium with M, M in equilibrium with N, and N in equilibrium with O. With increasing dehydration the kinetics were increasingly dominated by the reverse reactions. As before, fitting the data required the existence of two M species in series: L in equilibrium with M1 in equilibrium with M2 in equilibrium with N. The M1 in equilibrium with M2 reaction was greatly slowed at lower humidities. This step might be the switch for the unidirectional transfer of protons. With increasing dehydration recovery of BR occurred less and less via the N intermediate and increasingly via direct shunts from the two M species. As indicated earlier by electrical measurements with similarly dried bacteriorhodopsin films (Váró, G., and L. Keszthelyi, 1983. Biophys. J. 43:47-51). The latter are pathways not necessarily associated with net proton translocation.

The N intermediate of bacteriorhodopsin at low temperatures: Stabilization and photoconversion

In aqueous suspensions of purple membranes (pH 10.2, 0.4 M KCl) an intermediate having an absorption maximum at 570–575 nm (at −196°C) was produced by first heating the M intermediate up to −30°C and then stabilizing it by subsequent cooling to −60°C. We suggest that this species is the intermediate N (or P or R) found and characterized earlier near room temperature. Upon illumination at −196°C N is transformed into a bathochromically absorbing species K n which has an absorption maximum near 605 nm and an extinction 1.35 times that of N. This light reaction is photoreversible. The quantum yield ratio for the forward and back reaction is 0.18 ± 0.02. The maximum photo steady state concentration of K n is about 0.24. The N intermediate was also trapped in water suspensions of purple membranes at neutral pH and low salt concentration by illumination at λ > 620 nm during cooling. In addition to N another intermediate absorbing in the red (maximum at 610–620 nm) was accumulated in smaller amounts. It is not photoactive at 196°C and apparently is the O intermediate or a photoproduct of N.

Solid-state carbon-13 and nitrogen-15 NMR study of the low pH forms of bacteriorhodopsin

The visible absorption of bacteriorhodopsin (bR) is highly sensitive to pH, the maximum shifting from 568 nm (pH 7) to approximately 600 nm (pH 2) and back to 565 nm (pH 0) as the pH is decreased further with HCl. Blue membrane (lambda max greater than 600 nm) is also formed by deionization of neutral purple membrane suspensions. Low-temperature, magic angle spinning 13C and 15N NMR was used to investigate the transitions to the blue and acid purple states. The 15N NMR studies involved [epsilon-15N]lysine bR, allowing a detailed investigation of effects at the Schiff base nitrogen. The 15N resonance shifts approximately 16 ppm upfield in the neutral purple to blue transition and returns to its original value in the blue to acid purple transition. Thus, the 15N shift correlates directly with the color changes, suggesting an important contribution of the Schiff base counterion to the "opsin shift". The results indicate weaker hydrogen bonding in the blue form than in the two purple forms and permit a determination of the contribution of the weak hydrogen bonding to the opsin shift at a neutral pH of approximately 2000 cm-1. An explanation of the mechanism of the purple to blue to purple transition is given in terms of the complex counterion model. The 13C NMR experiments were performed on samples specifically 13C labeled at the C-5, C-12, C-13, C-14, or C-15 positions in the retinylidene chromophore. The effects of the purple to blue to purple transitions on the isotropic chemical shifts for the various 13C resonances are relatively small. It appears that bR600 consists of at least four different species. The data confirm the presence of 13-cis- and all-trans-retinal in the blue form, as in neutral purple dark-adapted bR. All spectra of the blue and acid purple bR show substantial inhomogeneous broadening which indicates additional irregular distortions of the protein lattice. The amount of distortion correlates with the variation of the pH, and not with the color change.

Transient conductivity change during the photocycle in purple membrane suspension

A transient conductivity change is detected in purple membrane (PM) suspension during the photocycle. This transient conductivity change is strongly dependent on the orientation of the PM. A.c. electric alignment of the PM suspension plays an important role in the detection of the conductivity change during a single excitation of the PM suspension. The transient conductivity change is analysed as a function of time and it is shown that the kinetics of this transient change are very similar to the kinetics of M and O state decay.