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At least 19 recordsLinked to original sources
PubMed1982

Location of the carboxyl terminus of bacteriorhodopsin in purple membrane.

Purple membrane samples have been prepared by trypsin digestion to have either 10 or 21 residues removed from the carboxyl terminus of the proteins. Electron diffraction of single membranes and x-ray diffraction of unoriented membrane pellets have been carried out on both these specimens and on native purple membranes. the main conclusion from this work is that the carboxyl terminus is almost entirely disordered, being free to take up many positions, and that its removal does not affect the packing in the crystal. The low resolution x-ray diffraction difference map may also suggest the approximate location of the carboxyl terminus.

Bacteriorhodopsins↗
PubMed1993

Aspartic acid-212 of bacteriorhodopsin is ionized in the M and N photocycle intermediates: an FTIR study on specifically 13C-labeled reconstituted purple membranes.

Purple membrane was regenerated from the denatured proteolytic (protease V8) fragments V-1 and V-2 of bacteriorhodopsin (BR), native membrane lipids, and all-trans-retinal. FTIR difference spectra of M and N intermediates of the reconstituted system are in close correspondence to those obtained from native BR. Asp-212 is the only internal aspartic acid in the V-2 fragment (helices F and G). Reconstituting a V-2 fragment from a [4-13C]Asp-labeled BR preparation with an unmodified V-1 fragment and vice versa have allowed us to assign IR bands to either Asp-212 or any of the remaining aspartic acids on V-1 (helices A-E). A carboxylate vibration at 1392 cm-1 has been identified in the M and N intermediates and assigned to Asp-212. Since no contribution of this residue to C = O stretches of protonated carboxyl groups was detected, Asp-212 must be ionized in light-adapted BR as well. The effect of [4-13C]Asp labeling of V-1 revealed a carboxylate vibration at 1385 cm-1 in light-adapted BR. Since Asp-96 and Asp-115 are protonated, this band is caused by Asp-85. All absorption changes of C = O stretches of protonated carboxyl groups are due to Asp residues on V-1. Correspondingly, the proton acceptor for Schiff base deprotonation in M is located on V-1, and must be Asp-85 (the only ionized Asp on V-1). The band assignments are compared with those reported for BR mutants, and the potential role of Asp-212 for proton translocation is discussed.

Aspartic Acid↗
PubMed1986

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange.

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.

Animals↗
PubMed1976

Kinetics and stoichiometry of light-induced proton release and uptake from purple membrane fragments, Halobacterium halobium cell envelopes, and phospholipid vesicles containing oriented purple membrane.

We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.

Bacteriorhodopsins↗
PubMed1978

Phospholipid substitution of the purple membrane. The stoichiometry of light-induced proton release by phospholipid-substituted purple membranes.

The method of Warren et al. (1974, Proc. Natl. Acad. Sci. U.S. 71, 622--626) was employed to substitute the polar lipids of the purple membrane of Halobacterium halobium by different phosphatidylcholine species. Substitution at pH 6.5 yields proteolipid complexes in the form of bent open sheets which have a protein to lipid phosphorus ratio similar to the natural membrane, i.e. about 1 : 10 (mol/mol). The extent of substitution increases with the length of the fatty acid chain of the phosphatidylcholine used. The spectral properties of bacteriorhodopsin are only slightly affected by substitution of 95% of the lipid, except that the photocycle is slowed down appreciably. Due to this slow rate the M412 intermediate of the cycle accumulates in the light. Associated with this accumulation is a net light-induced proton release, which proved insensitive to uncoupler. A comparison between the net proton release and the amount of M412 accumulated, studied as a function of pH, shows that no fixed stoichiometry exists between the two processes. Phospholipid substitution by egg phosphatidylcholine at pH 7.5 or by egg phosphatidylethanolamine leads to preparations of purple membrane with 15 or 25 mol of phospholipid per mol of bacteriorhodopsin, respectively. These preparations seem to consist of closed membrane structures. They take up protons in the light in an uncoupler-sensitive way.

Bacteriorhodopsins↗
PubMed2000

Structural determinants of purple membrane assembly.

The purple membrane is a two-dimensional crystalline lattice formed by bacteriorhodopsin and lipid molecules in the cytoplasmic membrane of Halobacterium salinarum. High-resolution structural studies, in conjunction with detailed knowledge of the lipid composition, make the purple membrane one of the best models for elucidating the forces that are responsible for the assembly and stability of integral membrane protein complexes. In this review, recent mutational efforts to identify the structural features of bacteriorhodopsin that determine its assembly in the purple membrane are discussed in the context of structural, calorimetric and reconstitution studies. Quantitative evidence is presented that interactions between transmembrane helices of neighboring bacteriorhodopsin molecules contribute to purple membrane assembly. However, other specific interactions, particularly between bacteriorhodopsin and lipid molecules, may provide the major driving force for assembly. Elucidating the molecular basis of protein-protein and protein-lipid interactions in the purple membrane may provide insights into the formation of integral membrane protein complexes in other systems.

Bacteriorhodopsins↗
PubMed1987

Bent membrane model of the purple membrane. Theoretical details and further experimental data.

Theoretical treatment of the bent membrane model of purple membrane is given. Beside the derivation of the formulae, the results of the numerical calculations concerning the angle dependence of the light scattering changes are presented. The paper contains also experimental data on the scattering changes during the photocycle at several scattering angles, which are in good accordance with the theory.

Bacteriorhodopsins↗
PubMed1983

Nonionic detergent effects on spectroscopic characteristics and the photocycle of bacteriorhodopsin in purple membranes.

Treatment of purple membranes isolated from Halobacterium halobium with nonionic detergents caused the following effects in addition to solubilizing bacteriorhodopsin: a blue shift of the absorption spectrum of bacteriorhodopsin; an increase in tryptophan fluorescence of about twofold; an enhanced rate of bleaching in the presence of hydroxylamine; and the disappearance of the negative exciton band in the CD spectrum at 600 nm. Crosslinking of purple membranes by glutaraldehyde can prevent solubilization of bacteriorhodopsin by detergents. However, only the effect on the CD spectrum by detergents can be inhibited by crosslinking of the purple membranes prior to detergent treatment. Photocycle kinetics studies revealed that the apparent pK of the slow component of the M412 decay was decreased by the detergent treatment while the rise time for M412 formation is accelerated two- to threefold. Crosslinking also did not prevent these effects. These results demonstrate that nonionic detergents, apart from their action to monomerize bacteriorhodopsin, also affect retinal-apoprotein interactions.

Bacteriorhodopsins↗
PubMed1980

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.

Bacteriorhodopsins↗
PubMed1996

Control of the integral membrane proton pump, bacteriorhodopsin, by purple membrane lipids of Halobacterium halobium.

Brief exposure of purple membrane (PM) to dilute Triton X-100 eliminates the actinic light effect on the relative amounts of fast M (Mf) and slow M (Ms) intermediates and alters the character and kinetics of the photocycle, without destroying the native BR trimers (Mukhopadhyay et al., 1994). Particular membrane lipids are removed during the Triton treatment, and adding back an extract of membrane lipids can repair most of the affected photocycle behavior (Dracheva et al., 1996). This paper defines conditions which are important in the reconstitution procedure, using a group of quantitative parameters which measure the extents of damage and repair. Circular dichroism in both the UV and visible ranges shows that Triton can disturb both the secondary structure of BR and its ability to polymerize into trimers. Whereas the damage to protein conformation could be reversed by lipids alone, the formation of trimers and recovery of normal photocycle behavior required both lipids and a high salt concentration.

Bacteriorhodopsins↗
PubMed1975

Improved isolation procedures for the purple membrane of Halobacterium halobium.

Techniques for purifying teh purple membrane of Halobacterium halobium are given. This purple membrane contains a chromoprotein with a retinal prosthetic group similar to rhodopsin, the chromprotein found in the visual systems of higher invertebrates and vertebrates. The described purple membrane isolation procedures yield a highly purified preparation as determined by transmitting electron microscopy and gel electrophoresis. Critical analysis of the absorption spectra of the purple membrane was also employed to establish criteria of purity for the preparation. The visible absorption spectra of the purified purple membrane preparation in buffer was found to have a maximum at 559 nm which shifted to 567 nm on light exposure. No indication of any spectral perturbation arising from bacterioruberin-containing membrane, the major contaminant in purple membrane preparations, was found. Furthermore, the ratio of protein aromatic amino acid absorbance at 280 nm to chromophore absorbance at 567 nm was found to be 1.5 in light-exposed preparations compared to the previously reported ratio of 2.3.-3 The decrease in the value of this ratio is also indicative of an increase in the purity of the purple membrane preparation.

Cell Fractionation↗
PubMed1991

Effects of volatile anesthetics on bacteriorhodopsin in purple membrane, Halobacterium halobium cells and reconstituted vesicles.

In this study, we have investigated effects of volatile anesthetics on absorption spectra, proton pumping activity and decay of photointermediate M of bacteriorhodopsin (bR) in differently aggregated states. Anesthetics used in this study are ether-type general anesthetics; enflurane and sevoflurane. The observed effects on bR depend not only on variety or concentration of anesthetics but also strongly on the aggregation state of bR molecules in the membrane. In purple membrane (PM), bR having maximum light absorption at 567 nm (bR567) is formed in the presence of sevoflurane or a small amount of enflurane, while a species absorbing maximally at 480 nm (bR480) is formed upon the addition of large amounts of enflurane. X-ray diffraction studies show that the former species maintains crystallinity of PM, but the latter does not. In reconstituted vesicles where bR molecules exist as monomer, even sevoflurane forms bR480. Flash photolysis experiments show that bR567 contains a shorter-lived M intermediate absorbing maximally at 412 nm in the photoreaction cycle than bR does and that bR480 contains at least two long-lived M intermediates which seem to absorb maximally near and at lower than 380 nm. The measurements of light-induced pH changes of the whole cells and of the reconstituted vesicles in the presence of the anesthetics indicate that bR567 has a enhanced proton pumping efficiency, while bR480 has a quite low or no activity. No significant difference was observed in the anesthetic action between two inversely pumping vesicles. These observations suggest that on the formation of bR480, anesthetics enter into the membrane and affect the protein-lipid interaction.

Anesthetics↗
PubMed1994

A comparison of the second harmonic generation from light-adapted, dark-adapted, blue, and acid purple membrane.

The second order nonlinear polarizability and dipole moment changes upon light excitation of light-adapted bacteriorhodopsin (BR), dark-adapted BR, blue membrane, and acid purple membrane have been measured by second harmonic generation. Our results indicate that the dipole moment changes of the retinal chromophore, delta mu, are very sensitive to both the chromophore structure and protein/chromophore interactions. Delta mu of light-adapted BR is larger than that of dark-adapted BR. The acid-induced formation of the blue membrane results in an increase in the delta mu value, and formation of acid purple membrane, resulting from further reduction of pH to 0, returns the delta mu to that of light-adapted BR. The implications of these findings are discussed.

Adaptation, Physiological↗
PubMed1996

Lipid-induced conformational changes of an integral membrane protein: an infrared spectroscopic study of the effects of Triton X-100 treatment on the purple membrane of Halobacterium halobium ET1001.

Exposure of purple membrane from Halobacterium halobium to sublytic concentrations of Triton X-100 results in significant changes in the bacteriorhodopsin (BR) photocycle (Mukhopadhyay et al., 1994). Infrared spectra of purple membrane samples exposed briefly to Triton indicate that this change in protein function accompanies the preferential release of purple membrane glycolipids and squalenes, an association of Triton with purple membrane, and a perturbation of specific lipid headgroup interactions within the membrane. Specifically, the bilayer alterations induced by Triton entail a disruption of lipid headgroup hydrogen bonding in addition to protein conformational changes involving a loss in beta-turn and alphaII-helical structures in BR. We propose that the purple membrane glycolipids and squalenes are critical for the normal functioning of the BR photocycle and that perturbations of these lipids cause the profound photocycle changes induced by exposure to Triton. Lipid reconstitution studies demonstrated that although several of the infrared spectral parameters characteristic of the structural changes induced by Triton were reversed, the photocycle characteristics of BR in native purple membrane were not regained. The observed changes in the vibrational spectra induced by lipid-mediated bilayer perturbations suggest a useful approach for clarifying structure-function relationships of intrinsic membrane proteins exhibiting transmembrane helices.

Bacteriorhodopsins↗