Single bacteriorhodopsin molecules revealed on both surfaces of freeze-dried and heavy metal-decorated purple membranes.

The flat sheets of the purple membrane from Halobacterium halobium contain only a single protein (bacteriorhodopsin) arranged in a hexagonal lattice. After freeze-drying at -80 degrees C (a method that is superior to air-drying), shadowing with tantalum/tungsten, and image processing, structural details on both surfaces are portrayed in the range of 2 nm. One surface is rough and lattice lines are clearly visible, whereas the other is smooth and the hexagonal order seems to be absent. The optical diffraction patterns, however, indicate a hexagonal lattice for both surfaces. In addition, these diffraction patterns are characteristic and easily distinguished. The orientation of the two surfaces was identified by silver decoration: partial condensation of silver on purple membranes enabled the smooth surface to be identified as the plasmatic and the rough surface as the exoplasmic surface. After image processing, the exoplasmic surface shows a triplet structure which exactly fits the projected structure determined by Unwin and Henderson (1975. Nature(Lond.). 257:28-32) at molecular resolution, whereas, on the plasmatic surface, four image details per unit cell are visible. Three of them match the arrangement of bacteriorhodopsin, whereas the fourth must be located over a lipidic array. Summarizing these results, it is possible to show the part of each single bacteriorhodopsin protein that is present in the surfaces of the purple membrane. By "shadowing" the membranes perpendicularly, we prove that these components of the surfaces are mainly portrayed by a decoration effect of the tantalum/tungsten condensate.

Direct observation of the femtosecond excited-state cis-trans isomerization in bacteriorhodopsin.

Femtosecond optical measurement techniques have been used to study the primary photoprocesses in the light-driven transmembrane proton pump bacteriorhodopsin. Light-adapted bacteriorhodopsin was excited with a 60-femtosecond pump pulse at 618 nanometers, and the transient absorption spectra from 560 to 710 nanometers were recorded from -50 to 1000 femtoseconds by means of 6-femtosecond probe pulses. By 60 femtoseconds, a broad transient hole appeared in the absorption spectrum whose amplitude remained constant for about 200 femtoseconds. Stimulated emission in the 660- to 710-nanometer region and excited-state absorption in the 560- to 580-nanometer region appeared promptly and then shifted and decayed from 0 to approximately 150 femtoseconds. These spectral features provide a direct observation of the 13-trans to 13-cis torsional isomerization of the retinal chromophore on the excited-state potential surface. Absorption due to the primary ground-state photoproduct J appears with a time constant of approximately 500 femtoseconds.

Interaction of purple membrane with solvents. I. Applicability of solubility parameter mapping.

We carried out spectral studies on the interaction between purple membrane fragments (isolated from Halobacterium halobium) and a series of different solvents, classified quantitatively according to their solubility parameters delta d, delta p, delta h. These represent the contribution of dispersion forces, polar forces, and hydrogen bonding, respectively, to the cohesive energy density of the solvent. Purple membrane fragments, kept in the dark, were suspended in each of the solvents as well as in binary mixtures of solvents, and the spectrum of the resulting suspension was recorded in the wavelength region 250-700 nm. The interaction of each solvent with the membrane fragments can be represented by a point on either a ternary diagram, where each of the three axes represents one of the solubility parameters, or a binary diagram, where one of the two axes is a combination of two of the solubility parameters (delta v = square root of delta d2 + delta p2 or delta a = square root of delta p2 + delta h2). In the former type of solvent map the contribution of each of the parameters is distinct but only their relative contributions are expressed. In the latter the absolute values of delta i are considered. In each of these modes of presentation an inner closed region is observed. The solvents inside its borders interact with bacteriorhodopsin with a resultant spectral change. Mixtures of solvents fit the maps according to their calculated delta values. Thus, a mixture of an apolar solvent with a highly polar solvent interacts with bacteriorhodopsin, even though each of these solvents alone does not.

Interaction of purple membrane with solvents. II. Mode of interaction.

Using the solubility parameter mapping technique (Eisenbach, M., Caplan, S.R. and Tanny, G (1979) Biochim. Biophys. Acta 554, 269-280) we studied spectroscopically the mode of interaction between the purple membrane of Halobacterium halobium and pure organic solvents or solvent mixtures. Although the interacting solvents formed a well-defined closed region in the interaction maps, mapping the modes of interaction did not reveal a closed region for each spectrally classifiable type. A suggested interpretation for this is that interaction with the purple membrane chromophore requires that a solvent (or solvent mixture) possess apolar groups in order to obtain access to the chromophore, together with a polar character and hydrogen-bonding capacity. The mode of interaction, however, is dependent on the specificity of the reactive group of the solvent for retinal, and this has nothing to do with membrane properties. We also examined the influence of the duration of the interaction and of illumination. Some solvents appeared to react more sluggishly than others, but no generalization in terms of the solubility parameter mapping was found, probably because the map describes thermodynamic rather than kinetic phenomena. The only effect of illumination was to enhance the reaction of some of these solvents. It did not change the solubility parameters of purple membrane.

Formation of the two-dimensional hexagonal lattice of bacteriorhodopsin in reconstituted brown membrane.

The aggregation state of reconstituted bacteriorhodopsin molecules in the brown membrane has been investigated by X-ray diffraction and CD spectra. It has been confirmed that reconstituted bacteriorhodopsin molecules form the hexagonal lattice spontaneously whereas bacterioopsin molecules do not.

The lipids of Halobacterium marismortui, an extremely halophilic bacterium in the Dead Sea.

The lipids of an extremely halophilic bacterium, Halobacterium marismortui, isolated from the Dead Sea, were found to contain 86% polar lipids and 14% non-polar lipids. Four major polar lipids were detected, all derivatives of 2,3-di-O-phytanyl-sn-glycerol: (1) a novel glycolipid, 2,3-di-O-phytanyl-1-O-[beta-D-glucopyranosyl-(1'-6')-O-alpha-D-mannopyranosyl-( 1'-2')-O-alpha-D-glucopyranosyl]-sn-glycerol (11 mol%); (2) phosphatidylglycerol (11 mol%); (3) phosphatidylglycerophosphate (62 mol%); (4) phosphatidylglycerosulfate (17 mol%). In addition, a minor glycolipid (less than 1 mol%) was detected and partially characterized. Trace levels of two other unidentified glycolipids and of two unidentified phospholipids were also detected. In contrast to Halobacterium cutirubrum and H. halobium, H. marismortui did not contain any detectable sulfated glycolipid but appeared to compensate for this deficit in sulfate by having a high content of phosphatidylglycerosulfate compared to that in H. cutirubrum. The number of negative changes per mol ionic lipid appeared to be about the same for both halophiles. The non-polar lipids in H. marismortui consisted mostly of squalenes, vitamin MK-8 and bacterioruberins with traces of beta-carotene, lycopene and retinal, as in H. cutirubrum.

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.

Bacteriorhodopsin (BR570) bathochromic band shift in an external electric field.

In dry films of bacteriorhodopsin-containing purple membranes from Halobacterium halobium the external electric field (10(4) -- 10(5) V . cm-1) induces the appearance of a product spectrally close to the initial intermediate of bacteriorhodopsin (BR) photochromic cycle (bathoform, K). This result and also preliminary data of the electret-thermal analysis of the preparations suggest that the dielectric polarization in chromophore-protein-lipid complexes might be an essential step of the primary stabilization of light energy in photo-bioenergetic processes.

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.

Thermodynamic studies of purple membrane.

Differential dilatometric and differential scanning calorimetric measurements have been made of purple membrane with an emphasis upon the temperature range 5 degrees C less than T less than 45 degrees C. The coefficient of thermal expansion alpha is about 7 X 10(-4)/Cdeg up to 30 degrees C and decreases at higher temperatures. The specific heat increases rapidly with temperature with absolute values in the range 0.30-0.45 cal/Cdeg per g. A nearly constant alpha juxtaposed with a rapidly increasing specific heat is similar to the properties of lipid bilayers in the gel phase and alkanes in the solid phase. This behavior is explained by the concept of hindered vibrations which would now appear to apply to at least one integral membrane protein. There may also be a small broad transition centered near 20-25 degrees C that would correspond to the melting of less than 25 degrees of freedom per bacteriorhodopsin molecule and associated lipids. Using our measured apparent specific volume the average thickness of purple membrane is calculated to be 43.5 A. The specific volume of interaction of lipids and proteins is estimated, using the amino acid sequence of bacteriorhodopsin and average amino acid volumes from structural studies of other proteins, to be about 11% of the specific volume of the purple membrane lipids or 4% of the volume of the bacteriorhodopsin protein. A positive volume of interaction is consistent with lipid-protein interactions being an important determinant of the thermodynamic properties of purple membrane.

Thermal denaturation and photochemistry of bacteriorhodopsin from Halobacterium cutirubrum as monitored by resonance Raman spectroscopy.

Resonance Raman studies of the thermal denaturation of bacteriorhodopsin from Halobacterium cutirubrum show that the N-retinylidenelysine moiety present in the chromophore is N-protonated. This corroborates an earlier suggestion of Lewis et al. ((1974) Proc. Natl. Acad. Sci. U.S., 71, 4462-4466). The widely differing excitation profiles of two -C=C- stretching modes are explained in terms of the light-initiated reaction cycle in the molecule. Glutaraldehyde fixation of bacteriorhodopsin has no effect on the intensity ratio of the two modes, suggesting that no large motion of the protein is necessary for the photoreaction cycle to occur.

The C-terminus of bacteriorhodopsin is a random coil.

The 21 amino acids which can be selectively removed from the carboxyl terminus of bacteriorhodopsin by proteolytic treatment are disordered in 2-dimensional arrays of the protein present in purple membranes. This C-terminal portion of the molecule may be involved in the efficiency and rate of light-driven proton uptake, although its presence is not required for pumping activity. In this study, the secondary structure of the C-terminus of bacteriorhodopsin has been determined by examining circular dichroism (CD) difference spectra derived from native and digested samples. In low ionic strength media, this part of the molecule appears to form a random coil-like structure. To examine if this structure is related to the structure found under the high ionic strength condition present in halobacteria, the CD spectra of native purple membranes in water and in 4 M salt solutions were compared. They were found to be identical, suggesting the conformation of the C-terminus in vivo may also be a random coil.

Light-dark adaptation of bacteriorhodopsin in triton-treated purple membrane.

Solubilization of purple membrane with Triton X-100 yields Triton micelles containing bacteriorhodopsin monomers. The absorption maximum of dark-adapted solubilized bacteriorhodopsin is blue-shifted to 549 nm. Light adaption increases the absorbance by 4% and shifts the absorption maximum to 553 nm, i.e., the extent of light adaptation is considerably less than in intact purple membrane. Extraction of dark-adapted bacteriorhodopsin in Triton yields a 13-cis- to all-trans-retinal ratio of 58 : 42 which changes after light adaptation to 38 : 62. It has been shown by Sperling et al. (Sperling, W., Carl, P., Rafferty, Ch.N. and Dencher, N.A. (1977) Biophys. Struct. Mech. 3, 79-94) that light adaptation in intact purple membrane occurs through a branching of the 13-cis photoreaction cycle, so that part of the pigment during each cycle crosses over into the all-trans photoreaction cycle. We explain the decreased extent of light adaptation in solubilized bacteriorhodopsin by assuming a significant back reaction from the all-trans to the 13-cis cycle. This assumption predicts a wavelength dependence of the extent of light adaptation, which is born out by experiment.

Light- and dark-adapted bacteriorhodopsin, a time-resolved neutron diffraction study.

Recently, neutron diffraction experiments have revealed well-resolved and reversible changes in the protein conformation of bacteriorhodopsin (BR) between the light-adapted ground state and the M-intermediate of the proton pumping photocycle (Dencher, Dresselhaus, Zaccai and Büldt (1989) Proc. Natl. Acad. Sci. USA 86, 7876-7879). These changes are triggered by the light-induced isomerization of the chromophore retinal from the all-trans to the 13-cis configuration. Dark-adapted purple membranes contain a mixture of two pigment species with either the all-trans- or 13-cis-retinal isomer as chromophore. Employing a time-resolved neutron diffraction technique, no changes in protein conformation in the resolution regime of up to 7 A are observed during the transition between the two ground-state species 13-cis-BR and all-trans-BR. This is in line with the fact that the conversion of all-trans BR to 13-cis-BR involves an additional isomerization about the C15 = N Schiff's base bond, which in contrast to M formation minimizes retinal displacement and keeps the Schiff's base in the original protein environment. Furthermore, there is no indication for large-scale redistribution of water molecules in the purple membrane during light-dark adaptation.

Isolation and characterization of C50-carotenoid pigments and other polar isoprenoids from Halobacterium cutirubrum.

The polar acetone-soluble lipids of Halobacterium cutirubrum were found to contain (in addition to the previously reported vitamin MK-8 and retinal) neo-bacterioruberin U, bacterioruberin, monoanhydrobacterioruberin, bis-anhydrobacterioruberin, an isomer of geranylgeraniol (with one internal cis-isoprene residue), 2,3,-di-O-phytanyl-sn-glycerol and two unidentified polar isoprenoids. All compounds were isolated in pure form by column and thin-layer chromatography, quantitated and characterized by their visible, ultraviolet, infrared, proton magnetic resonance and mass spectra and the spectra of their acetyl or silyl derivatives and/or dehydrated products.

The preparation of lipid-depleted bacteriorhodopsin.

Bacteriorhodopsin, the protein of the purple membrane of Halobacterium halobium, was freed to the extent of 90-95% from the natural membrane lipids without loss of function. The residual lipid corresponded to less than 1 mol/mol of bacteriorhodopsin. Delipidation was achieved by treatment of the purple membrane with a mixture of the detergent dimethyldodecylamine oxide and sodium chloride. The detergent was removed by dialysis or by sucrose density gradient centrifugation. Analysis of the lipids removed and those still bound to bacteriorhodopsin was facilitated by the use of purple membrane preparations labelled with 35S, 32P, or 14C. The composition of the residual lipids associated with bacteriorhodopsin was similar to that of the total lipid in the purple membrane.

Fluorescent labeling of bacteriorhodopsin: implications for helix connections.

Purple membrane from Halobacterium halobium was reacted with dansyl (5-dimethylamino-1-naphthalenyl fluorescent labels that have specificity for different protein side chains of bacteriorhodopsin. Dansyl chloride was found to react primarily with Lys-41. Dansyl hydrazine was coupled, with water-soluble carbodiimide, to Glu-74 and/or Asp-85, which was the major modified site after papain-cleavage of the carboxyl-terminal 17 amino acids. Fluorescence energy transfer was used to probe the proximity of the modified sites to the retinal chromophore of bacteriorhodopsin. The dansyl group on Lys-41 was greater than 2.99 nm from retinal, while the dansyl group on Glu-74/Asp-85 was greater than 2.10 nm from retinal. Information available on the location of retinal in the transmembrane profile and probable surface locations of the fluorescent labels was combined with the energy transfer results to calculate distances projected in the plane of the membrane. The projected distances to retinal were 1.64 nm (Lys-41) and 1.65 nm (Gly-74). These measurements, combined with many other labeling experiments that have been reported, restrict the number of likely helix-connection models to only three: EDCABGF, FEDCBAG and FGEABDC (in the nomenclature of Engelman et al. (1980) Proc. Natl. Acad. Sci. USA 77, 2023-2027).

Effect of lipid-protein interaction on the color of bacteriorhodopsin.

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.

Measuring changes in membrane thickness by scanning tunneling microscopy.

We investigated the feasibility of using the scanning tunneling microscope (STM) as a morphometric tool to measure the thickness of biomembranes. Planar monolayers of oriented purple membrane (PM) were prepared, nitrogen-dried or freeze-etched, and coated with metal. PM thickness was quantified by STM and transmission electron microscopy. STM calibration and the effect of contamination-mediated surface deformation on measurements of PM thickness were evaluated. The thickness of PM attached to mica and glass and the effect of papain on PM thickness were also examined. The apparent thickness of enzymatically modified PM increased after papain treatment. The mean thickness of both nitrogen-dried PM on mica and freeze-etched PM on glass was 4.6 nm. After papain treatment PM thickness on mica increased to 4.8 nm and on glass to 5.4 nm. These results demonstrate that STM analysis of metal-coated planar membrane monolayers can be used to measure changes in average membrane thickness at sub-nanometer resolution.

Isoelectric forms of bacteriorhodopsin from Halobacterium halobium.

Isoelectric focusing was performed on bacteiorhodopsin isolated from Halobacterium halobium, R1 strain, solubilized in Nonidet P-40, on sucrose density gradient stabilized columns. When 560 nm absorbance was monitored, four forms of bacteriorhodopsin were observed, having isoelectric points (pI) of: A. 3.93; B, 4.43; C, 5.03; D, 5.49. Instability of some of the isoelectric forms during the very process of electrofocusing was observed. When focused over a period of 6 days, the relative abundance of the forms changed although their pI values remains constant. Refocusing of the isolated forms A or B led to the production f form C. The latter species was stable to refocusing. Form B was unstable to storage either as an aqueous suspension of the purple membrane or as a detergent extract. Each of the forms had the absorption spectrum typical for bacteriorhodopsin and each showed identical patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.