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PubMedDate not supplied

Surface dynamics of the integral membrane protein bacteriorhodopsin.

Recently, there has been great interest in determining the three-dimensional structures of membrane proteins, particularly bacteriorhodopsin, for which a variety of possible folding arrangements have been suggested. In this paper we present nuclear magnetic resonance (NMR) spectra of deuterated bacteriorhodopsin, and use the data to help interpret the various suggested bacteriorhodopsin folding patterns. The results strongly indicate that (1) a membrane surface (+/- 1 residue) may be defined by NMR in bacteriorhodopsin; (2) all amino acids inside the surface are essentially crystalline; (3) all amino acids outside the surface (surface residues) in bacteriorhodopsin are highly mobile on the time scale of the 2H NMR experiments; (4) NMR data may be used to help evaluate the various structural models that have been proposed; (5) aggregation of purple membrane sheets may lead to an immobilization of the surface residues.

Amino Acid Sequence↗
PubMed1981

Transmembranous incorporation of photoelectrically active bacteriorhodopsin in planar lipid bilayers.

Various methods to incorporate bacteriorhodopsin in black lipid membranes are reported. Both purple membrane patches and monomeric bacteriorhodopsin were used as starting material. The incorporation of bacteriorhodopsin into planar lipid bilayers was achieved by the following methods. (i) Purple membrane patches were transferred from water to solutions of lipids in n-alkanes. Black membranes were formed from such organic suspensions. (ii) Lipid layers containing solvent and purple membranes were spread on an air/water interface. These layers were used to form planar bilayers. (iii) Vesicles containing purple membranes or monomeric bacteriorhodopsin were spread on an air/water interface and, from the resulting layer, bilayers were formed. On illumination, steady-state photocurrents were observed in all three cases, indicating that these methods lead to functional transmembranous integration of the protein in the planar black lipid membrane. The influence of an applied electric field on the pumping process was studied on membranes formed by using method i. At approximately 200 mV, the photocurrent tends to zero. Furthermore, it was possible to make planar lipid bilayers photoelectrically active by adding vesicles containing monomeric bacteriorhodopsin to the bathing solution. Because, in this case, only transient photocurrents were observed, it can be concluded that the vesicles are attached to but not fused with the black lipid membrane.

Bacteriorhodopsins↗
PubMed1981

Bacteriorhodopsin: partial sequence of mRNA provides amino acid sequence in the precursor region.

mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis.

Amino Acid Sequence↗
PubMed1995

Protein conformational changes during the bacteriorhodopsin photocycle. A Fourier transform infrared/resonance Raman study of the alkaline form of the mutant Asp-85-->Asn.

Bacteriorhodopsin is a light-driven proton pump, which undergoes a photocycle consisting of several distinct intermediates. Previous studies have established that the M-->N step of this photocycle involves a major conformational change of membrane embedded alpha-helices. In order to further investigate this conformational change, we have studied the photocycle of the high pH form of the mutant Asp-85-->Asn (D85Nalk). In contrast to wild type bacteriorhodopsin, D85Nalk has a deprotonated Schiff base and a blue-shifted absorption near 410 nm, yet it still transports protons in the same direction as wild type bacteriorhodopsin (Tittor, J., Schweiger, U., Oesterhelt, D. and Bamberg, E. (1994) Biophys. J., 67, 1682-1690). Resonance Raman spectroscopy of D85Nalk and D85Nalk regenerated with retinal labeled at the C-15 position with deuterium reveals the existence of an all-trans configuration of the chromophore. Fourier transform infrared difference spectroscopy shows that the photocycle of this light-adapted form involves similar events as the wild type bacteriorhodopsin photocycle including the M-->N protein conformational change. These results help to explain the ability of D85Nalk to transport protons and demonstrate that the M-->N conformational change can occur even in the photocycle of an unprotonated Schiff base form of bacteriorhodopsin.

Asparagine↗
PubMed1999

Bacterioopsin-triggered retinal biosynthesis is inhibited by bacteriorhodopsin formation in Halobacterium salinarium.

Factors regulating retinal biosynthesis in halobacteria are not clearly understood. In halobacteria, events leading to the biosynthesis of bacteriorhodopsin have been proposed to participate in stringent regulation of retinal biosynthesis. The present study describes a novel approach of in vivo introductions of mRNA and membrane proteins via liposome fusion to test their role in cellular metabolism. Both the bacterioopsin-encoding mRNA and the liposome-encapsulated bacterioopsin (apoprotein) are independently introduced in spheroplasts of the purple membrane-negative strain Halobacterium salinarium that initially contain neither bacterioopsin nor retinal. Isoprenoid analyses of these cells indicate that the expression/presence of bacterioopsin triggers retinal biosynthesis from lycopene, and its subsequent binding to opsin generates bacteriorhodopsin. When bacteriorhodopsin and excess retinal were independently introduced into spheroplasts of purple membrane-negative cells, the introduction of bacteriorhodopsin resulted in an accumulation of lycopene, indicating an inhibition of retinal biosynthesis. These results provide direct evidence that the formation of bacterioopsin acts as a trigger for lycopene conversion to beta-carotene in retinal biosynthesis. The trigger for this event does not lie with either transcription or translation of the bop gene. It is clearly associated with the folded and the membrane-integrated state of bacterioopsin. On the other hand, the trigger signaling inhibition of retinal biosynthesis does not lie with the presence of excess retinal but with the correctly folded, retinal-bound form, bacteriorhodopsin.

Amino Acid Sequence↗
PubMed2000

brp and blh are required for synthesis of the retinal cofactor of bacteriorhodopsin in Halobacterium salinarum.

Bacteriorhodopsin, the light-driven proton pump of Halobacterium salinarum, consists of the membrane apoprotein bacterioopsin and a covalently bound retinal cofactor. The mechanism by which retinal is synthesized and bound to bacterioopsin in vivo is unknown. As a step toward identifying cellular factors involved in this process, we constructed an in-frame deletion of brp, a gene implicated in bacteriorhodopsin biogenesis. In the Deltabrp strain, bacteriorhodopsin levels are decreased approximately 4.0-fold compared with wild type, whereas bacterioopsin levels are normal. The probable precursor of retinal, beta-carotene, is increased approximately 3.8-fold, whereas retinal is decreased by approximately 3.7-fold. These results suggest that brp is involved in retinal synthesis. Additional cellular factors may substitute for brp function in the Deltabrp strain because retinal production is not abolished. The in-frame deletion of blh, a brp paralog identified by analysis of the Halobacterium sp. NRC-1 genome, reduced bacteriorhodopsin accumulation on solid medium but not in liquid. However, deletion of both brp and blh abolished bacteriorhodopsin and retinal production in liquid medium, again without affecting bacterioopsin accumulation. The level of beta-carotene increased approximately 5.3-fold. The simplest interpretation of these results is that brp and blh encode similar proteins that catalyze or regulate the conversion of beta-carotene to retinal.

Bacteriorhodopsins↗
PubMed2002

Irreversible photobleaching of bacteriorhodopsin in a high-temperature intermediate state.

The photo-intermediate state of bacteriorhodopsin is a metastable state that spontaneously transforms to the ground state over the energy barrier of a local minimum. As the recovery of the photocycle to the ground state and irreversible photobleaching to the denatured state may occur from the same local energy minimum, depending on the temperature, the structural stability of bacteriorhodopsin under illumination at high temperature was measured in order to study the intra- and inter-molecular interactions that contribute to the recovery of the ground state. Visible CD spectra of bacteriorhodopsin began to change at 60 degrees C from a bilobed to positive type in accordance with an appearance of an absorption peak at 470 nm. Irreversible photobleaching, the light-induced denaturation, also started to occur at 60 degrees C, suggesting some correlation between irreversible photobleaching and the structural change to the high-temperature intermediate state. However, bacteriorhodopsin in the dark was stable up to 70 degrees C, suggesting that there is some additional factor that lends structural stability to bacteriorhodopsin in the dark. The contribution of protein-protein interactions to stability is discussed on the basis of the difference in the denaturation behaviors between light and dark conditions.

Bacteriorhodopsins↗
PubMed1977

Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin.

Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis.

Adenosine Triphosphatases↗
PubMed1992

1H-15N-NMR studies of bacteriorhodopsin Halobacterium halobium. Conformational dynamics of the four-helical bundle.

Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1-71), B1 (residues 1-155) and BP2 (residues 163-231) were investigated. By this procedure, all observed 1H-15N cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross-peaks from four (C, D, E and F) helical bundles (79-189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.

Amino Acid Sequence↗
PubMed1996

delta psi-mediated signalling in the bacteriorhodopsin-dependent photoresponse.

It has been shown previously that the proton-pumping activity of bacteriorhodopsin from Halobacterium salinarium can transmit an attractant signal to the bacterial flagella upon an increase in light intensity over a wide range of wavelengths. Here, we studied the effect of blue light on phototactic responses by the mutant strain Pho8l-B4, which lacks both sensory rhodopsins but has the ability to synthesize bacteriorhodopsin. Under conditions in which bacteriorhodopsin was largely accumulated as the M412 bacteriorhodopsin photocycle intermediate, halobacterial cells responded to blue light as a repellent. This response was pronounced when the membrane electric potential level was high in the presence of arginine, active oxygen consumption, or high-background long-wavelength light intensity but was inhibited by an uncoupler of oxidative phosphorylation (carbonyl cyanide 3-chlorophenylhydrazone) and was inverted in a background of low long-wavelength light intensity. The response to changes in the intensity of blue light under high background light was asymmetric, since removal of blue light did not produce an expected suppression of reversals. Addition of ammonium acetate, which is known to reduce the pH gradient changes across the membrane, did not inhibit the repellent effect of blue light, while the discharge of the membrane electric potential by tetraphenylphosphonium ions inhibited this sensory reaction. We conclude that the primary signal from bacteriorhodopsin to the sensory pathway involves changes in membrane potential.

Acetates↗
PubMedDate not supplied

The interaction between halogenated anaesthetics and bacteriorhodopsin in purple membranes as examined by intrinsic ultraviolet fluorescence.

In the presence of halogenated general anaesthetics such as enflurane and halothane, the spectral properties of the bacteriorhodopsin pigment contained in the purple membranes of Halobacterium halobium are strongly modified. It is reversibly transformed into a red-coloured species absorbing maximally at 480 nm, at the expense of its characteristic 570-nm absorption band. The ultraviolet fluorescence of bacteriorhodopsin has been used to probe the structural modifications that are reflected by this spectral change. Our results show that they are very small and do not perturb the energy transfer dynamics which take place between the aromatic amino acid residues and the retinyl chromophore. The fluorescence properties of anaesthetic-treated bacteriorhodopsin are dominated by the quenching properties of the halogenated hydrocarbon, which are obvious even at anaesthetic concentrations under those needed to induce a spectral change in the bacteriorhodopsin chromophore. This does not rule out direct interaction between anaesthetics and bacteriorhodopsin, but it indicates that the chromophoric site might well not be their primary target.

Bacteriorhodopsins↗
PubMed2001

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.

Bacteriorhodopsins↗
PubMed1992

Protein changes associated with reprotonation of the Schiff base in the photocycle of Asp96-->Asn bacteriorhodopsin. The MN intermediate with unprotonated Schiff base but N-like protein structure.

The difference Fourier transform infrared spectrum for the N intermediate in the photoreaction of the light-adapted form of bacteriorhodopsin can be recorded at pH 10 at 274 K (Pfefferlé, J.-M., Maeda, A., Sasaki, J., and Yoshizawa, T. (1991) Biochemistry 30, 6548-6556). Under these conditions, Asp96-->Asn bacteriorhodopsin gives a photoproduct which shows changes in protein structure similar to those observed in N of wild-type bacteriorhodopsin. However, decreased intensity of the chromophore bands and the single absorbance maximum at about 400 nm indicate that the Schiff base is unprotonated, as in the M intermediate. This photoproduct was named MN. At pH 7, where the supply of proton is not as restricted as at pH 10, Asp96-->Asn bacteriorhodopsin yields N with a protonated Schiff base. The Asn96 residue, which cannot deprotonate as Asp96 in wild-type bacteriorhodopsin, is perturbed upon formation of both MN at pH 10 and N at pH 7. We suggest that the reprotonation of the Schiff base is preceded by a large change in the protein structure including perturbation of the residue at position 96.

Asparagine↗
PubMed1992

A bacteriorhodopsin analog reconstituted with a nonisomerizable 13-trans retinal derivative displays light insensitivity.

With the aim of preparing a light-insensitive bacteriorhodopsin-like pigment, bacterio-opsin expressed in Escherichia coli was treated in phospholipid-detergent micelles with the retinal analog II, in which the C13-C14 trans-double bond cannot isomerize due to inclusion in a cyclopentene ring. The formation of a complex with a fine structure (lambda max, 439 nm) was first observed. This partially converted over a period of 12 days to a bacteriorhodopsin-like chromophore (ebR-II) with lambda max, 555 nm. An identical behavior has been observed previously upon reconstitution of bleached purple membrane with the analog II. Purification by gel filtration gave pure ebR-II with lambda max, 558 nm, similar to that of light-adapted bacterio-opsin reconstituted with all-trans retinal (ebR-I). Spectrophotometric titration of ebR-II as a function of pH showed that the purple to blue transition of bacteriorhodopsin at acidic pH was altered, and the apparent pKa of Schiff base deprotonation at alkaline pH was lowered by 2.4 units, relative to that of ebR-I. ebR-II showed no light-dark adaptation, no proton pumping, and no intermediates characteristic of the bacteriorhodopsin photocycle. In addition, the rates of reaction with hydroxylamine in the dark and in the light were similar. These results show, as expected, that isomerization of the C13-C14 double bond is required for bacteriorhodopsin function and that prevention of this isomerization confers light insensitivity.

Bacteriorhodopsins↗
PubMed1987

Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F.

To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.

Amino Acid Sequence↗
PubMed1982

Immunological probes for bacteriorhodopsin. Identification of three distinct antigenic sites on the cytoplasmic surface.

We have prepared site-specific immunological reagents to study the orientation and surface topography of the integral membrane protein bacteriorhodopsin. Monoclonal and polyclonal antibodies with strong affinity for antigenic determinants on proteolytic and cyanogen bromide fragments of bacteriorhodopsin have been isolated and characterized. Three distinct antibody binding sites have been identified on the cytoplasmic surface of bacteriorhodopsin. The first due is readily accessible in native bacteriorhodopsin and lies close to the COOH terminus. This binding site is lost when only three amino acid residues are removed from the COOH terminus. The second site, which is also near the COOH terminus, is located approximately within the 17 COOH terminal amino acid residues. The third site is in the fragment that comprises Tyr-83 to Met-118 and is probably contained in the short loop connecting the third and fourth helices. The use of COOH terminus-specific antibodies in determination of the orientation of bacteriorhodopsin molecules in the Halobacterium halobium membrane confirms the earlier conclusion that the COOH terminus is on the cytoplasmic side.

Amino Acid Sequence↗
PubMed1983

Introduction and characterization of amber mutations in the bacteriorhodopsin gene.

A fusion between the genes for bacteriorhodopsin and beta-galactosidase was constructed on a multicopy plasmid, pXB/Gal 101. The fusion gene, containing the bacteriorhodopsin gene fused upstream from the beta-galactosidase gene, was under the control of tandem lipoprotein and lac gene promoters. When expressed in Escherichia coli the fusion protein retained beta-galactosidase activity. Mutations in the fusion gene were produced by passage of pXB/Gal 101 through the E. coli mutator strain mut D5. Amber mutations were then selected by examining the loss of the lac+ phenotype imparted by the fusion protein to lac- E. coli cells. Amber mutations occurring within the bacteriorhodopsin gene were localized by replacing the beta-galactosidase region of each mutant plasmid with a beta-galactosidase region which was known to be unmutated. Precise localization of the mutations was achieved first by sizing the prematurely terminated peptides produced by the mutant plasmids in in vitro coupled transcription-translation reactions, and secondly by DNA sequence analysis. Six amber mutants in the gene for bacteriorhodopsin were characterized in this way. One of these was a transversion mutation at a lysine codon; the other five were all transition mutations at tryptophan codons, codons 10, 12, 80, 86, and 137 of the bacteriorhodopsin sequence.

Amino Acid Sequence↗
PubMed1993

Hydrophobic amino acids in the retinal-binding pocket of bacteriorhodopsin.

The hydrophobic amino acids Met-20, Val-49, Ala-53, Met-118, Gly-122, and Met-145 are believed to be among the amino acids that form the retinal-binding pocket in bacteriorhodopsin. We have now replaced the above amino acids, one at a time, and report on the effects of these replacements (M20A/E, V49A/L, A53G, M118A/E, G122C, and M145A/E) on the properties of bacteriorhodopsin. With the exception of Met-20, replacements at all of the other positions resulted in (i) altered rates of in vitro chromophore formation that ranged from 20-fold faster to 45-fold slower than wild-type bacteriorhodopsin, (ii) blue shifts in the visible spectra of up to 80 nm, and (iii) caused changes in the retinal isomer compositions of the mutant chromophores. Specific effects were also observed in the photocycles of the Met-118, Met-145, and Val-49 mutants, suggesting that these 3 amino acids have important roles in light transduction by bacteriorhodopsin. These results are discussed together with previous studies on the effects of amino acid replacements in the retinal-binding pocket of bacteriorhodopsin.

Amino Acid Sequence↗