Light-induced conductivity changes in purple membrane suspensions.

Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different event: a small fast increase in conductivity (tau approximately 2 ms) followed by a slower and larger decrease in this parameter (tau = 70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31 degrees C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occurring in the system.     

Light-induced conductivity changes in purple membrane suspensions

Small light-induced changes in the conductivity of light-adapted purple membrane suspended in strong electrolyte solutions were detected. The method used involved modulated light and a phase sensitive detector and it allowed us to detect accurately changes as small as 0.0001% in the conductivity of the suspension. The light-induced conductivity changes turned out to be composed of at least two different events: a small fast increase in conductivity (t ∼ 2 ms) followed by a slower and larger decrease in this parameter (Τ=70 ms-80 ms). The effects of pH and temperature on these changes were studied. Both events reached maximal values around neutral pH and approached zero at both high and low pH's. Heating the suspension decreased the photoconductivity change and Arrhenius plots of the data showed breaks around 31‡ C. It is suggested that the conductivity changes reflect changes in the surface charge of the membrane and can be used to follow the kinetics of the conformational changes occuring in the system.     

Light-induced conductivity changes of purple membrane suspensions in strong electrolytes

Measurements have been made of the modulated light-induced changes in conductivity and the associated relaxation times of bacteriorhodopsin in a variety of strong electrolytes, both unbuffered and buffered. The effects of pH and temperature variation have been studied as well as the effect of adding valinomycin. Two relaxation times can be distinguished: a fast lifetime associated with protonation-deprotonation, and a slow lifetime associated with ion binding. The ion-binding effects appear to be cation specific.     

Light-induced currents from oriented purple membrane: II. Proton and cation contributions to the photocurrent

The sign of B2, the micro-second component of the photocurrent from oriented purple membrane, is that of positive charge moving away from the purple membrane in the direction of proton release. B2 could be due to internal dipole or proton movement, proton release, or metal cation release. We found that the waveform of B2 is virtually insensitive to changes in the salt concentration as long as it is >40 mM KCl, >5 mM CaCl₂, or >0.5 mM LaCl₃. However, below these limits, B2's apparent rate of decay increases as the salt concentration decreases without any change in the initial amplitude. This salt dependence suggests that B2 is due to a positive charge, either a metal cation or a proton, moving from the membrane into the solution. That the positive charge is not a metal cation is suggested by the waveform of B2 remaining unchanged upon replacing the cations both in solution and in the binding sites of the purple membrane. Direct evidence that the positive charge movement is due to protons was obtained by examining the correlation of B2 with the proton dependent processes of bacteriorhodopsin in buffers and dyes. Based on these observations, we suggest that most, if not all, of the intrinsic B2 component of the photocurrent at moderate salt concentration is due to proton release.

The photocurrents from purple membranes whose surface potential has been reduced by delipidation or chemical modification of carboxyl groups with methyl esters were found to be only modestly changed. This suggests that the salt effect is not through its modulation of the surface potential. Rather, we propose that in low salt B2 represents the sum of a proton release from the surface of the purple membrane and a second current component, due to cations moving back towards the membrane, which is only important in low salt. The cation counter current is induced by proton release which creates a transient uncompensated negative charge on the membrane.

     

Non-proton ion release in purple membrane.

Large conductivity changes have been measured during the photocycle of bacteriorhodopsin in purple membrane. These phenomena were explained as being due to the occurrence of large-scale non-proton ion release. Here we show that these conductivity changes do not appear if the purple membrane is immobilized. We propose an alternative hypothesis that explains the presence of conductivity change in suspensions and their absence in gels, as well as several related effects suggesting that the observed conductivity changes are due to alteration of the polarizability of purple membrane during the photocycle.     

Photoselection and circular dichroism in the purple membrane.

The transient dichroic ratio D = delta A parallel/delta A perpendicular has been measured in the visible absorption region of bacteriorhodopsin in purple membrane by a flash photolysis method. D is found to be wavelength independent throughout the visible absorption band, and reaches a maximum value of 2.75 +/- 0.15 on reduction of the excitation intensity. This value is close to that expected for a single nondegenerate transition dipole moment and is incompatible with the strong exciton coupling model used to explain circular dichroism (CD) spectrum of purple membrane. A time-dependent analysis of the exciton interaction and consideration of the coupling strength suggests an explanation of these observations. It is concluded that excitation interaction between retinals in purple membrane is of the weak or very weak type defined by Förster.     

Flash spectroscopy of purple membrane.

Flash spectroscopy data were obtained for purple membrane fragments at pH 5, 7, and 9 for seven temperatures from 5 degrees to 35 degrees C, at the magic angle for actinic versus measuring beam polarizations, at fifteen wavelengths from 380 to 700 nm, and for about five decades of time from 1 microsecond to completion of the photocycle. Signal-to-noise ratios are as high as 500. Systematic errors involving beam geometries, light scattering, absorption flattening, photoselection, temperature fluctuations, partial dark adaptation of the sample, unwanted actinic effects, and cooperativity were eliminated, compensated for, or are shown to be irrelevant for the conclusions. Using nonlinear least squares techniques, all data at one temperature and one pH were fitted to sums of exponential decays, which is the form required if the system obeys conventional first-order kinetics. The rate constants obtained have well behaved Arrhenius plots. Analysis of the residual errors of the fitting shows that seven exponentials are required to fit the data to the accuracy of the noise level.     

Light-induced currents from oriented purple membrane: I. Correlation of the microsecond component (B2) with the L-M photocycle transition

When irradiated, purple membrane from Halobacterium halobium oriented in a polyacrylamide gel produces a photocurrent. The correlation of the microsecond component (B2) of the photocurrent with the L-M optical transition was studied. It was found that the lifetimes of B2 and the L-M transition are identical over the entire pH range from 2.4 to 11.0 when measured in high salt (>5 mM CaCl₂ or >40 mM KCl). Changing the temperature from 10 to 35°C, or replacing the H₂O with D₂O maintains this correlation. The amplitude of B2 and the L-M transition are also correlated over the pH range where both of them can be represented as a single exponential. At high pH (>8), three exponentials were required to fit both the optical and photocurrent signals. Two of them are the previously described fast and slow components of M formation, but a new intermediate with a very fast lifetime, 0.3 μs, was observed both in absorption (λ = 410 nm) and photocurrent measurements. The lifetimes of all three were found to be pH independent. This would exclude models for the L to M portion of the photocycle that explained its complex pH-dependent behavior as being due to a single pH-dependent rate constant. The area of B2, which is proportional to the number and the distance the charge moved during the transition, is almost constant from pH 5.0 to pH 8.0. It decreases to almost zero at pH 2.0 and pH 10.6 with pKs at 2.8 and 9.1. Because B2 is thought to normally reflect proton release from the membrane, this suggests at very low and high pH the photocycle does not pump protons. The pK at high pHs for the formation of the nonpumping photocycle is probably related to the formation of a new photocycle featuring the fast rising form of M.     

Energy transfer in the purple membrane of Halobacterium halobium.

The absorption spectrum of the primary photoproduct (the bathoproduct, or K) of the purple membrane protein (PM) at-196 degrees C has a maximum at 628 nm and an extinction coefficient of 87,000. Knowing the absorption spectrum allowed us to calculate the quantum efficiencies for PM to K and K to PM conversion at -196 degrees C. Direct measurements of these quantum yeilds at -196 degrees C gave 0.33 +/- 0.05 and 0.67 +/- 0.04, respectively. Determination of relative quantum efficiencies for PM to K and K to PM conversion by analysis of the absorption spectra of several photostationary-state mixtures of PM and K at -196 degrees C, however, gave wavelength-dependent quantum efficiencies that appear to be greater than 1. These anomolous results can be readily explained in terms of energy transfer from PM to K within the trimer clusters of pigment molecules which exist in the purple membrane. A model for such a transfer predicts an efficiency of energy transfer from PM to K of about 43%.     

Electric dichroism in the purple membrane of Halobacterium halobium.

Aqueous suspensions of fragments of the purple membrane of Halobacterium halobium are exposed to short electric field pulses. The relaxation kinetics of the induced dichroism are studied as a function of environmental factors such as temperature, medium viscosity, and treatment of the membranes with glutaraldehyde and dimethylsulfoxide. The data indicate that the alignment of the retinyl chromophore is due to orientation of the whole membrane fragments with their planes parallel to the electric field, as well as to an intramembrane orientation of bacteriorhodopsin molecules (or of a part of such molecules). Wavelength effects on the dichroic ratio show that weak, out of (membrane) plane components contribute to the chromophore spectrum on the red side (lambda greater than 560 nm) of the main (alpha) absorption band as well as the range of the beta band (lambda less than 480 nm). The former effect is attributed to exciton interactions, while the latter is assigned to the contribution of a transition to the lowest 1Ag+ state ("cis" band). It is also concluded that the transition moment along the short (kappa) axis, in the plane of the polyene molecule, has a substantial component perpendicular to the membrane plane.     

Melittin-regenerated purple membrane

We have investigated the character of melittin-regenerated purple membrane. Adding melittin to blue membrane causes the color transition and partial regeneration of the photocycle and the proton pump. The reconstitution of bacteriorhodopsin by melittin is proved to be charge-dependent. In studying the location of melittin binding on the blue membrane, we suggest that melittin anchors on the membrane through both hydrophobic and electrostatic interactions. The electro-static interaction is dominant. The binding sites for the electrostatic interaction should be on the surface of the membrane.     

Immuno-atomic force microscopy of purple membrane.

The atomic force microscope is a useful tool for imaging native biological structures at high resolution. In analogy to conventional immunolabeling techniques, we have used antibodies directed against the C-terminus of bacteriorhodopsin to distinguish the cytoplasmic and extracellular surface of purple membrane while imaging in buffer solution. At forces > or = 0.8 nN the antibodies were removed by the scanning stylus and the molecular topography of the cytoplasmic purple membrane surface was revealed. When the stylus was retracted, the scanned membrane area was relabeled with antibodies within 10 min. The extracellular surface of purple membrane was imaged at 0.7 nm resolution, exhibiting a major and a minor protrusion per bacteriorhodopsin monomer. As confirmed by immuno-dot blot analysis and sodium dodecyl sulfate-gel electrophoresis, labeling of the purple membrane was not observed if the C-terminus of bacteriorhodopsin was cleaved off by papain.     

Reaction of the purple membrane with a carbodiimide.

Purple membrane was reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.5 and 8.0. At pH 4.5, the reaction yields cross-linked bacteriorhodopsin. The cross-linking is inhibited by pretreatment of the membrane with papain, or by the presence of carbohydrazide or glycine ethyl ester in the reaction mixture. The product of the pH 8.0 reaction is not cross-linked, but it displays altered properties. Two measures of photochemical activity (light-induced change in proton binding (delta h) and decay of photointermediate M) show changes indicative of slowed proton uptake. The delta h is increased by ethyl dimethylaminopropylcarbodiimide. This increase is unaffected by pretreatment of the membrane with papain, and it is not reversed by NH2OH. However, the reaction is inhibited by millimolar concentrations of CaCl2. The altered delta h is not apparent in detergent-solubilized membranes. Ethyl dimethylaminopropylcarbodiimide does not appear to cause a large alteration in the membrane surface charge, as measured by Ca2+ binding. We conclude that (1) at acid pH, ethyl dimethylaminopropylcarbodiimide can be used for cross-linking or for attachment of specific probes to the C-terminal region of bacteriorhodopsin, and hence to the cytoplasmic side of the purple membrane, and (2) at alkaline pH, ethyl dimethylaminopropylcarbodiimide reacts at a diffent type of site and appears to inhibit the proton pump.     

Synthetic pigment analogues of the purple membrane protein.

Nonphysiological analogues of retinal have been shown to form pigments in reactions with the apoprotein of the purple membrane of Halobacterium halobium. Both the all-trans and 13-cis isomers of a retinal analogue, having an elongated chain with an extra double bond, formed pigments. Unlike the native all-trans and 13-cis retinal1-based pigments, the new pigments were not interconvertible with each other and were unstable against hydroxylamine. When incorporated into phospholipid vesicles, they showed no proton pumping activity upon illumination. The ability of the extended-length retinal to form pigments contrasts with its nonreactivity with opsin (apoprotein of rhodopsin), suggesting a less stringent binding site for the purple membrane chromophore. All-trans retinal2 also combined with bleached purple membrane to form a blue pigment absorbing at ca. 590 nm. Like the native purple membrane, the blu membrane showed proton pumping activity upon illumination in phospholipid vesicles.     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )