Electric field effects in bacteriorhodopsin.

Exposure of aqueous suspensions of fragments of the purple membrane of Halobacterium halobium to electric field pulses leads to transient linear dichroism phenomena. The effects are interpreted in terms of field-induced alignments of the bacteriorhodopsin chromophore. Two observed relaxation times (tau) are attributed to rotation of the whole membrane fragments (tau s approximately 100 ms), and to a much faster reorientation of the chromophore within membrane (tau f approximately 260 microns).     

Conformational changes of bacteriorhodopsin detected by Fourier transform infrared difference spectroscopy

We report the first Fourier transform infrared difference spectra of purple membrane. Evidence is presented that alterations in the vibrations of both the retinylidene chromophore and the protein groups of bacteriorhodopsin associated with photocycling can be detected. This method provides a new tool for probing the conformational changes occurring in bacteriorhodopsin during the proton pump cycle.     

Structural and spectroscopic characteristics of bacteriorhodopsin in air-water interface films

A suspension of purple membrane fragments in a solution of soya phosphatidyl-choline in hexane is spread at an air-water interface. Surface pressure and surface potential measurements indicate that the membrane fragments and lipids organize at the interface as an insoluble film. Electron microscopy of shadow-cast replicas of the film reveal that in the bacteriorhodopsin to soya PC weight ratio range of 2:1 to 10:1, these films consist of nonoverlapping membrane fragments which occupy approximately 35% of the surface area and are separated by a lipid monolayer. Furthermore, the membrane fragments are oriented with their intracellular surface towards the aqueous subphase. Nearly all the bacteriorhodopsin molecules at the interface are spectroscopically intact and exhibit visible spectral characteristics identical to those in aqueous suspensions of purple membrane and in intact bacteria. In addition, bacteriorhodopsin in air-dried interface films show spectral changes upon dark-adaptation and upon flash illumination similar to those observed in aqueous suspensions of purple membrane, but with slower kinetics. The kinetics of the spectral changes in interface films can be made nearly the same as in aqueous suspension by immersing the films in water.     

Circular dichroism study of bacteriorhodopsin-lipid interaction

Delipidated bacteriorhodopsin purified from purple membrane of H. halobium was reconstituted with the circular dichroism active phospholipid. The observed circular dichroism spectra in the 450-700 nm region characteristic of bacteriorhodopsin showed the temperature dependence characterized by a midpoint at ca. 45 degrees C and this spectral change showed the disaggregation of bacteriorhodopsin trimer to monomer. The circular dichroism spectra in the 250-400 nm region characteristic of the azo chromophore of phospholipid exhibited a remarkable temperature dependence synchronized with the disaggregation of bacteriorhodopsin, suggesting that a large proportion of the phospholipid is present as boundary lipid.     

Proteolysis and flash photolysis of bacteriorhodopsin in purple membrane fragments

Pronase treatment of aqueous suspensions of purple membrane fragments from H. halobium leads to the cleavage of bacteriorhodopsin. The protein fragments remaining in the membrane after treatment with relatively small concentrations of enzyme (2% w/w) in normal daylight range in molecular weight from 20,000-21,000 daltons, indicating that cleavage occurs mainly near the extremities of the protein chain. At higher enzyme concentrations the relative amounts of protein fragments having smaller molecular weight increase. Generally, the relative loss of retinal chromophore is larger than that of protein and thus the retinal binding site seems to be located near one of the chain ends that is cleaved off by enzyme.Irradiation with white light during the time of proteolysis (at both low and high enzyme concentrations) results in extensive cleavage, so that under certain conditions no high molecular weight components can be detected in SDS-polyacrylamide gels. It, therefore, appears that parts of the bacteriorhodopsin chain become more exposed to enzyme digestion when the purple membrane is illuminated.Enzyme treated aqueous purple membrane fragment suspensions still show photocycle activity. The main consequence of proteolysis is a pronounced appearance of biphasicity in the decay of M₄₁₂ and the regeneration of bR₅₇₀. Simultaneously the yield of O₆₆₀ is reduced. As with untreated purple membrane, the correlation between the rates of decay of M₄₁₂ and regeneration of bR₅₇₀ is greatest when the yield of O₆₆₀ is lowest.     

Characterization of the binding of valinomycin to bacteriorhodopsin

Circular dichroism spectroscopy has been used to investigate the binding of valinomycin to bacteriorhodopsin in purple membrane suspensions. Addition of valinomycin to purple membrane suspensions obtained from Halobacterium halobium causes the circular dichroism spectrum to shift from an aggregate spectrum to one resembling a monomer spectrum, indicating a loss of chromophore-chromophore interactions. By observing the spectral change upon titration of valinomycin, an apparent dissociation constant of 30–40 M for valinomycin binding was determined. Kinetics of dark adaptation for valinomycin-treated purple membrane are comparable to those for monomeric bacteriorhodopsin. Centrifugation studies demonstrate that valinomycin-treated purple membrane sediments the same as untreated purple membrane suspensions. These results are consistent with a model in which valinomycin binds specifically to bacteriorhodopsin without disrupting the purple membrane fragments.Abbreviations BR bacteriorhodopsin - CD circular dichroism - Tricine N-[tris-(hydroxymethyl) methyl] glycine     

Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.     

Bacteriorhodopsin: the effect of bilayer thickness on 2D-array formation, and the structural re-alignment of retinal through the photocycle

From our earlier extensive protein-lipid reconstitution studies, the conditions under which bacteriorhodopsin forms organised 2D arrays in large unilamellar vesicles have been established using freeze-fracture electron microscopy. In a background bilayer matrix of phosphatidylcholine (diC(14:0)), the protein can form arrays only when the anionic purple membrane lipid, phosphatidylglycerol phosphate (or the sulphate derivative) is present. Here we have now extended this work to investigate the effect of bilayer thickness on array formation. Phosphatidylcholines with various chain lengths (diC(12:0), diC(14:0) and diC(16:0)) and which form bilayers of well defined bilayer thickness, have been used as the matrix into which bacteriorhodopsin, together with minimal levels (c. 4-10 lipids per bacteriorhodopsin) of diphytanyl phosphatidyl-glycerol phosphate, has been reconstituted. Arrays are formed in all complexes and bhickness appears only to alter the type of array formed, either as an orthogonal or as an hexagonal array. Secondly, we have previously deduced the entire conformation of retinal within the bacteriorhodopsin binding pocket in oriented purple membrane fragments. Using solid state deuterium NMR of the specifically deutero-methylated retinal labelled at each of the methyl positions in the molecule, the C-CD(3) bond vectors of the chromophore have been resolved to +/- 2 degrees . The ring conformation is 6-S-trans, but the polyene chain is slightly curved when in the protein binding site. Here, we describe studies on the protein in both the ground state and the trapped M(412)-state of the photocycle, to show that the orientation of the central methyl group (C(19)) on the polyene chain, which is at 40 degrees +/- 1 degrees with respect to the membrane normal, only changes its orientation by approximately 4 degrees upon 13-cis-isomerization. Thus, it is the Schiff base end of the chromophore which moves upon light incidence acting as a local switch on the protein in the photocycle, whilst the ring end of the chromophore moves rather less.     

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.     

Dimeric-like kinetic cooperativity of the bacteriorhodopsin molecules in purple membranes.

The kinetics of the absorption changes accompanying the photocycle of bacteriorhodopsin (BR) strongly depend on the intensity of the exciting short laser pulse. The decrease in the flash intensity dependence of the M kinetics after different extents of bleaching of the purple membranes by hydroxylamine proves the existence of a cooperative interaction between the photocycling BR molecules. The yield of the slow component of the M decay (M(s)) is a quadratic function of the extent of the fraction cycling. The slope of the relative weight of M(s) versus the fraction cycling is 0.5. This slope indicates a dimeric-like cooperative interaction, although the structural units of the purple membranes are the trimers of the BR molecules. For the most probable cooperative mechanism an asymmetric trimeric interaction is suggested, which accounts for the apparently dimeric features. A photocycling molecule may influence only one of its two neighbors in the trimer. From this asymmetric feature a deformative interaction is expected to be the cooperative mechanism, which would be an allosteric regulating mechanism in the purple membrane.     

Light-induced reorientation in the purple membrane.

Reorientation of bacteriorhodopsin in the native purple membrane was studied by time-resolved linear dichroism spectroscopy (TRLD) over the millisecond time regime. The time responses observed in TRLD are distinctly different from the isotropic transient absorption (TA) at wavelengths in the range 550-590 nm, where the bacteriorhodopsin ground state absorbs. In contrast, the TA and TRLD responses have nearly identical time dependence at 410 and 690 nm, where the intermediates M and O, respectively, principally contribute. These results demonstrate ground-state bacteriorhodopsin reorientation triggered by the photocycle. The TRLD and TA data are analyzed to test models for reorientational motion. Rotational diffusion of ground-state bacteriorhodopsin cannot account for the details of the data. Rather, the results are shown to be consistent with a reversible reorientation of "spectator" (nonexcited) members of the bacteriorhodopsin trimer in the purple membrane in response to the photocycling member of the trimer. This response may be associated with cooperativity in the trimer.     

Electric birefringence study of the purple membrane of Halobacterium halobium

An electric birefringence study was carried out on aqueous suspensions of the purple membrane of Halobacterium halobium. In addition to the characterization of both native and modified membrane samples, the dependence of electric birefringence on pH and ionic strength was also investigated. The results indicate that purple membrane shows electric birefringence at a field strength as low as 200 V/cm. The permanent dipole moment and polarizability ranged from 20,500 debyes and 1.01 × 10^?14 cm³ for a purple membrane concentration of 0.40 mg/mL to 41,000 debyes and 2.05 × 10^?14 cm³ for a concentration of 0.80 mg/mL. It was also found that removal of the retinyl group of bacteriorhodopsin substantially decreases but does not eliminate the electric birefringence of the membrane. The solubilization of the membrane by Triton X-100, however, completely abolishes the electric birefringence. These experiments indicate that there is an interaction between adjacent bacteriorhodopsin molecules within the purple membrane via the retinyl chromophore moiety that builds up the permanent dipole moment. They also suggest that there are two types of response when purple membrane suspensions are placed in an electric field. One is an alignment of the disk-shaped particles with the field. The other is a stacking of the particles following their alignment by the electric field, which is promoted by the induced dipole moment.     

Thermodynamic properties of purple membrane

We measured the density, expansivity, specific heat at constant pressure, and sound velocity of suspensions of purple membrane from Halobacterium halobium and their constituent buffers. From these quantities we calculated the apparent values for the density, expansivity, adiabatic compressibility, isothermal compressibility, specific heat at constant pressure, and specific heat at constant volume for the purple membrane. These results are discussed with respect to previously reported measurements on globular proteins and lipids. Our data suggest a simple additive model in which the protein and lipid molecules expand and compress independently of each other. However, this simple model seems to fail to describe the specific heat data. Our compressibility data suggest that bacteriorhodopsin in native purple membrane binds less water than many globular proteins in neutral aqueous solution, a finding consistent with the lipid surround of bacteriorhodopsin in purple membrane.     

Restricted motion of photoexcited bacteriorhodopsin in purple membrane containing ethanol.

The molecular motion of retinal within the purple membrane was investigated by flash-induced absorption anisotropies with or without ethanol. In the absence of ethanol, the measured anisotropies at several wavelengths exhibited almost the same slow decay. This slow decay was attributed to only the rotation of purple membrane sheet itself in the aqueous suspension. In the presence of ethanol, however, we observed the wavelength-dependent anisotropies. The fluidity of the purple membrane, investigated with a fluorescence anisotropy method, was increased by the addition of ethanol. These facts indicated that the characteristic motion of bacteriorhodopsin is induced in perturbed purple membrane with ethanol. The data analysis was performed, taking account of the overlapping of absorption from ground-state bacteriorhodopsin and photointermediates. The results showed that the rotational motion of photointermediates within the membrane was more restricted than that of nonexcited bacteriorhodopsin. The addition of ethanol facilitated the rotation of nonexcited protein, whereas it did not significantly affect the motion of photointermediates. The restricted motion of photointermediates is probably caused by a conformational change in them, which may hinder the rotation of monomer protein and/or induce the interaction between photointermediate and neighboring proteins.     

Spectral heterogeneity in protein fluorescence of bacteriorhodopsin: evidence for intraprotein aqueous regions

By the use of derivative spectral analysis, the broad tryptophan (Trp) fluorescence emission from aqueous suspensions of bacteriorhodopsin in its native purple membrane may be resolved into contributions from buried, surface, and exposed residues. Addition of glycerol produces a progressive enhancement of the fluorescence yield to a limiting value at about 70% v/v glycerol. Glycerol enhancement of fluorescence is also observed for monomeric Trp, and a good correlation exists between this effect and literature estimates of the fractional degree of Trp exposure in nine globular proteins. The estimate of fractional Trp exposure in bacteriorhodopsin from this correlation (50 +/- 15%) is in agreement with the value obtained by spectral differentiation and also by modified Stern-Volmer curves for quenching by water-soluble acrylamide. The absence of significant quenching by Tb(III) or Eu(III) ions, which may be expected to bind to the purple membrane surface, shows that the exposed Trp residues are in contact with water in intraprotein regions of the membrane and may be the first direct evidence for a transmembrane aqueous channel by which protons are actively transported during the bacteriorhodopsin photochemical cycle.     

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.     

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.     

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.     

Effect of fluorescamine modification of purple membranes on exciton coupling and light-to-dark adaptation

Purple membranes of $\text{Halobacterium}\text{,}\text{halobium}$ were modified with fluorescamine. At pH 8.8, with a molar ratio of fluorescamine to bacteriorhodopsin of 170, about 6 residues of lysine were modified while the arginines were not affected at all. Except for the appearance of the fluorescamine peak at 394 nm and some broadening of the chromophore peak at 570 nm, the absorption spectrum of bacteriorhodopsin was not significantly changed after modification. After fluorescamine modification, circular dichroism studies indicated loss of exciton coupling between bacteriorhodopsin molecules in the purple membrane. Rotational diffusion studies suggested enhanced mobility of the chromophore after modification. However, the spectral changes accompanying the light-to-dark adaptation of purple membranes were not prevented by fluorescamine modification. The implications of these findings are that exciton coupling between neighboring bacteriorhodopsin molecules in the purple membrane is not required for light-to-dark adaptation.     

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements.

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.