THE pH DEPENDENCE OF TRANSIENT CHANGES IN THE CURVATURE OF THE PURPLE MEMBRANE*

Abstract– We have observed transient changes in the curvature of purple membrane fragments upon photoexcitation as a function of the pH of the suspending medium. The room temperature suspensions have low ionic strengths, and the bending is observed by changes in the scattered light at 320 nm. The photoexcitation is from a 20 ns laser pulse at 532 nm. We use a simple first-order approximation to subtract any changes in the scattered light associated with transient absorption changes during the photocycle. The resultant scattering curves are then fitted to the sum of three fundamental bending processes. Each process has an exponential rise, an exponential decay and an amplitude. We model two of the processes as transient forces correlated with the charge motion during the photocycle. The third process is probably cuased by local changes in the pH as the protein pumps protons. However, this is not proved rigorously. Additional experiments are proposed.     

FURTHER CHARACTERIZATION OF ANESTHETIC-TREATED PURPLE MEMBRANES

Abstract— The absorption maximum of bacteriorhodopsin is shifted from 568 nm to 480 nm when halogenated volatile anesthetics (enflurane; halothane) are added to purple membranes. Analysis of the rate of formation of this new species upon addition of the anesthetic and of the back-formation of native bacteriorhodopsin upon its removal indicate that in purple membranes, the dark-adapted chromophore is much less reactive than its light-adapted counterpart. Lipid-soluble molecules thus have a lower accessibility to the dark-adapted chromophore.
In addition, activity of the 480 nm bacteriorhodopsin was investigated. Flash and steady-state photolysis experiments reveal that this blue shifted chromophore has full photochemical activity. It has a meta-intermediate absorbing maximally at 380 nm. The photocycle ofBR–480 is mainly characterized by a slow decay of the "O" intermediate, enabling the direct observation of the branching reaction between the "M" intermediate and the parentBR–480 pigment.     

All-trans to 13-cis retinal isomerization in light-adapted bacteriorhodopsin at acidic pH

The flash photolysis kinetic spectra of the intermediate M(412) of bacteriorhodopsin were monitored during the process of acid titration. In the light-adapted state, the maximum peak amplitude of M(412) absorbance of bacteriorhodopsin decreased (pK(a)=3.40+/-0.05) as the pH decreased from 7.3 to 1.9. In the dark-adapted state, the maximum peak amplitude of M(412) absorbance of bacteriorhodopsin increased as the pH decreased from 6.9 to 4.1, and then decreased (pK(a)=2.85+/-0.05) as the pH dropped to 2.1. These different trends in the change in the maximum peak amplitude suggested that not only the transition of purple membrane to blue membrane had taken place in both light and dark-adapted states, but also the fraction of all-trans-bR had changed during the acid titration. The pH-dependent absorption changes at 640 nm of bacteriorhodopsin in both light- and dark-adapted states were also observed. The pK(a)-values of the purple-to-blue transition were 3.80+/-0.05 in light-adapted state and 3.40+/-0.05 in dark-adapted state, respectively. According to Balashov's method, the fraction of all-trans-bR was assayed as the pH decreased. All these results indicated that the purple-to-blue transition of light-adapted bacteriorhodopsin was accompanied by an all-trans to 13-cis retinal isomerization at acidic pH.     

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 Ob̊ transition.     

PICOSECOND KINETICS and A MODEL FOR THE PRIMARY EVENTS OF BACTERIORHODOPSIN

The picosecond absorption kinetics of light adapted bacteriorhodops in (BR) are reported for water and glycerol-water suspensions of purple membrane from Halobacieriwn hulobium at different temperatures (77-300 K) and pH (5-10). It is shown that a photoproduct of BR, called P-BR (pseudo-bacteriorhodopsin). is responsible for the 16 ± 2 ps relaxation lifetime observed under steady state irradiation (i.e. with a train of ps pulses) at room temperature. At 77 K the absorption recovery lifetime is 62 ± 5 ps in good agreement with previous fluorescence lifetime studies. The observed signal is very sensitive to the sample flow rate and at the highest flow rates a fast absorbance increase (≤ 2 ps) is observed at λ > 620 nm. while at 576 nm a similarly fast absorption recovery alter bleaching is observed. These results imply that the reaction BR → K-BR occurs within 2 ps. In order to explain the simultaneous formation of P-BR and K-BR at 77 K. a model for the primary events including a traus-cis isomerization and a protein relaxation about the chromophore is suggested. The same model can also be used in explaining the simultaneous formation of batho- and hypsorhodopsin in some rhodopsins. Finally, a scheme for the last steps in the photocycle of bacteriorhodopsin including protonation, protein relaxation and cis-trans isomerization is proposed     

THE PHOTOREACTION OF THE DEIONIZED FORM OF THE PURPLE MEMBRANE INVESTIGATED BY FTIR DIFFERENCE SPECTROSCOPY

Abstract— Deionization of the purple membrane of Halobacterium halobium shifts the visible absorption maximum from 570 to 605 nm and inhibits proton transport. FTIR-difference-spectra of this blue membrane at 280 K reveal that the retinal chromophore adopts a 13 -cis and all -trans geometry in a light dependent ratio. In contrast to purple membrane the 13-cu isomer forms much faster in the dark. The all- trans component produces an L-intermediate which can be stabilized at 170 K. Spectral characteristics are similar to normal L. including comparable changes of internal aspartic acids of the opsin. However, stronger changes in the amide-I absorption are observed. IR bands of the chromo-protein states are assigned to retinal normal modes by the use of bacteriorhodopsin regenerated with'C-labeled retinals.     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

Thermochromism of bacteriorhodopsin and its pH dependence

Purple membranes (PMs), which consist of the photochromic membrane protein bacteriorhodopsin (BR) and lipids only, show complex thermochromic properties. Three different types of reversible temperature-dependent spectral transitions were found, involving spectral states absorbing at 460, 519, and 630 nm. These thermochromic absorption changes were analyzed in the range from 10 to 80 degrees C. In dependence on the bulk pH value, hypsochromic or bathochromic shifts in the BR absorption spectra are observed in BR gels as well as in BR films. The thermochromic changes between both purple and blue or purple and red were quantified in the CIE color system. The molecular changes causing these effects are discussed, and a model is presented in terms of intramolecular protonation equilibriums. The thermochromic properties of BR may be of interest in applications like security tags, as this feature may complement the well-known photochromic properties of BR.     

FLASH-INDUCED FAST CHANGE IN SURFACE POTENTIAL AND FLUIDITY OF PURPLE MEMBRANE STUDIED BY SPIN LABEL METHOD

Abstract— Flash-induced changes in surface potential and fluidity of purple membrane were studied by a spin label method. Changes in surface potential and fluidity were monitored by observing the distribution of charged and uncharged spin labels between the purple membrane and the aqueous phase.
On flash illumination, a transient hyperpolarization of the surface potential and a transient fluidization of the membrane hydrophobic region are induced. The former may probably reflect the proton movement near the purple membrane surface, while the latter may result from photo-induced conformational changes of bacteriorhodopsin.
The two events are different in time course. The relationships between the two events, and the formation and decay of the intermediates of bacteriorhodopsin in the photoreaction cycle are discussed.     

D96N mutant bacteriorhodopsin immobilized in sol-gel glass characterization

The D96N mutant form of bacteriorhodopsin (BR) purple membrane fragments isolated from the bacteriumHalobacterium salinarium has been immobilized by entrapment in sol-gel glass. The protein was characterized for M state decay rate at different temperatures and pH values. Bleaching efficiency and absorbance maxima vs pH were also determined. The kinetic effects of triethanolamine and diethanolamine were also examined. Results indicated that the immobilized BR was affected in a manner similar to the mutant BR in aqueous suspension. Addition of guanidine, however, caused the immobilized BR to show kinetic parameters more closely related to the wild-type protein than the D96N mutant control. Samples of the aqueous suspension were characterized for particle size and particle size distribution. Dried samples of the immobilized BR were analyzed by field emission microscopy and BET to characterize both the purple membrane fragments and the sol-gel pore characteristics.     

LASER PHOTOLYSIS OF THE PURPLE MEMBRANE OF Halobacterium halobium IN THE PHOTOSTATIONARY STATE: THE PHOTOBRANCHING PROCESS FROM THE O640 INTERMEDIATE

Abstract The photobranching process from the O₆₄₀ intermediate (O) in the photocycle of bacteriorhodopsin was studied. The O form accumulated with continuous wave visible light (390–800 nm) irradiation of the acidic (pH 3.9–6.0) purple membrane of Halobacterium halobium at 22°C. The photocycle of O via an L-like (or N-like) intermediate was driven by 630 nm pulsed light. The newly found intermediate has an absorption band in the 450–560 nm region. The "green-light"-absorbing pigment, tentatively called G₅₂₀, was converted to O with a time constant of (1.2 ± 0.2) ms. No M-like species was found in the cycle. The quantum yield of the cycle was estimated to be 0.30 ± 0.15.     

TIME-RESOLVED FOURIER TRANSFORM INFRARED SPECTROSCOPY OF THE BACTERIORHODOPSIN MUTANT TYR-185→E: ASP-96 REPROTONATES DURING O FORMATION; ASP-85 AND ASP-212 DEPROTONATE DURING O DECAY

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.     

LIGHT-INDUCED PROTON DISSOCIATION AND ASSOCIATION IN BACTERIORHODOPSIN

Abstract— Light-induced proton release and uptake by acetylated and unmodified bacteriorhodopsin were measured. Bacteriorhodopsin, when illuminated, shows a net proton release at neutral and alkaline pH's, but in acidic pH, it shows an uptake of protons. In the presence of high concentrations of guanidine hydrochloride, light caused only proton release even in acidic pH and the maximum extent of the release was one proton per bacteriorhodopsin molecule around pH 8.
Acetylation of bacteriorhodopsin caused no alteration in the absorption spectrum of purple complex (bR₅₇₀) and M₄₁₂-intermediate, but decreased the decay rate of the M₄₁₂-intermediate. Light-induced release of protons was not observed even in neutral pH values, and only the proton uptake was noticed by acetylated purple membrane fragments. In high concentrations of guanidine hydrochloride, no proton uptake or release by illumination was observed. Vesicles were reconstituted from acetylated purple membrane. These vesicles had almost no ability for light-induced proton transport. The role of amino group(s) in light-induced proton release and transport through the purple membrane is discussed.     

THE TWO CONSECUTIVE M SUBSTATES IN THE PHOTOCYCLE OF BACTERIORHODOPSIN ARE AFFECTED SPECIFICALLY BY THE D85N AND D96N RESIDUE REPLACEMENTS

The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to 400 nm, suggesting that this species in turn is affected by D85. These results are consistent with the suggestion that M1 and M2 are pre-switch and post-switch states, respectively.     

RESONANCE RAMAN SPECTRA OF THE "BLUE" and THE REGENERATED "PURPLE" MEMBRANES OF Halobacterium halobium

Abstract— We have obtained the resonance Raman spectra of the deionized form of the purple membrane, the so called blue membrane, as well as the purple membrane regenerated by titrating the blue membrane with either Na+, Ca²+ or La³+. All types of regenerated purple membrane have identical Raman spectra which are virtually indistinguishable from the native light-adapted bacteriorhodopsin spectrum. On the other hand, Raman data for the blue membrane suggest that it consists of essentially two pigment forms with absorption maxima around 605 and 570 nm and containing 13-cis and all-trans isomeric configurations of the chromophore. This is consistent with our chromophore extraction results which reveal that the blue membrane consists of 30% 13-cir and 70% all-trans chromophore.     

pH-dependent bending in and out of purple membranes comprising BR-D85T

The light-driven proton pump bacteriorhodopsin (BR) embedded in a purple membrane (PM) from Halobacterium salinarum undergoes a series of conformational changes while transporting a proton from the cytoplasmic to the extracellular side over the course of the so-called photocycle. Wild-type BR variant D85T, where aspartic acid 85 is replaced by threonine, allows for the study of structural intermediates of this photocycle that are formed in a light-dependent manner in the wild-type and in thermal equilibrium by tuning the pH of the D85T purple membrane suspension. Especially the last and least studied O-intermediate of the photocycle of bacteriorhodopsin has caught recent attention. First AFM images of D85T under acidic conditions resembling wild-type BR under physiological conditions in the O-photocycle-intermediate are presented. Bacteriorhodopsins embedded in the strongly bent purple membranes were analyzed by single molecule force spectroscopy (SMFS) providing the first single molecule force spectra of BR in the O-intermediate. SMFS was further employed to determine the absolute sign of membrane curvature. Complementary electrostatic force microscopy (EFM) was performed to support PM side discrimination and determination of the bending direction. Bending of PM-D85T was analyzed in more detail providing further insight into the structure-function relationship of the bacteriorhodopsin proton pump as well as PM behaviour at the solid-liquid junction. Findings reported here are of general interest to the field of chemomechanical transducers.     

The study of terbium regenerated bacterirhodopsin

Bacteriorhodopsin (bR) is the only retinal-contain- ing protein in the purple membrane of Halobacterium halobium[1]. Upon illumination, the protein undergoes a photocycle and pumps protons across the cell mem-brane[2,3]. It has been found that well-washed…     

Trapping and spectroscopic identification of the photointermediates of bacteriorhodopsin at low temperatures

Light-driven transmembrane proton pumping by bacteriorhodopsin occurs in the photochemical cycle, which includes a number of spectroscopically identifiable intermediates. The development of methods to crystallize bacteriorhodopsin have allowed it to be studied with high-resolution X-ray diffraction, opening the possibility to advance substantially our knowledge of the structure and mechanism of this light-driven proton pump. A key step is to obtain the structures of the intermediate states formed during the photocycle of bacteriorhodopsin. One difficulty in these studies is how to trap selectively the intermediates at low temperatures and determine quantitatively their amounts in a photosteady state. In this paper we review the procedures for trapping the K, L, M and N intermediates of the bacteriorhodopsin photocycle and describe the difference absorption spectra accompanying the transformation of the all-trans-bacteriorhodopsin into each intermediate. This provides the means for quantitative analysis of the light-induced mixtures of different intermediates produced by illumination of the pigment at low temperatures.     

THE WHITE MEMBRANE OF CRYSTALLINE BACTERIOOPSIN IN HALOBACTERIUM HALOBIUM STRAIN R1mW AND ITS CONVERSION INTO PURPLE MEMBRANE BY EXOGENOUS RETINAL

Abstract— A new strain isolated from Halobacterium halobium designated R₁mW, contained negligible amounts of isoprenoid pigments, had a yellowish white color due to respiratory pigments and showed no proton movement in response to light. However, addition of all-trans-retinal converted R₁mW into purple cells. Formation of both halorhodopsin and bacteriorhodopsin was indicated by induction of light-dependent proton uptake and release, respectively. Both haloopsin and bacterioopsin were thus postulated to be present in R₁mW. Electron micrographs of freeze-fractured cytoplasmic membranes revealed patches in a hexagonal array of trimeric particles, comparable to the purple membrane structure. These white membrane patches were isolated by procedures similar to those for the purple membrane. The white membrane's buoyant density was about 1.18 g/m/, and its main component migrated on sodium dodecylsulfate polyacrylamide gels at the same rate as bacteriorhodopsin. The white membrane showed only a small absorption peak at ～410nm due to contaminating respiratory pigments and a strong absorption at around 275 nm and shorter wavelengths. The white membrane was thus considered to be mainly composed of bacterioopsin, which was readily converted into bacteriorhodopsin by an addition of all-trans-retinal. The absorption and CD spectra of the white membrane were measured before and after addition of retinal. The molar extinction coefficient of dark-adapted bacteriorhodopsin formed was determined to be 53000M^?1 cm^?1 at 560 nm from retinal binding studies. The CD spectrum of the white membrane was negligible in the visible region but showed several bands assigned to aromatic and backbone structures in the UV region. Retinal addition caused considerable changes in the spectrum, yielding the CD spectrum of crystalline purple membrane bacteriorhodopsin. The white membrane thus appears to be a preparation suitable for structure-function studies of bacteriorhodopsin.