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At least 19 recordsLinked to original sources
PubMed2002

Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin, bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues.

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.

Adaptation, Physiological↗
PubMed1991

Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.

Bacteriorhodopsins↗
PubMed1994

Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin. 1. Proton migration along the surface of bacteriorhodopsin micelles and its delayed transfer from surface to bulk.

The kinetics of the light-induced release and uptake of protons was monitored with the optical pH-indicator fluorescein covalently bound to various sites on the extracellular and cytoplasmic surfaces of bacteriorhodopsin. Selective labeling was achieved by reacting (iodoacetamido)fluorescein with the single cysteine residues in bacteriorhodopsin introduced at the desired positions by site-directed mutagenesis. All measurements were performed with bacteriorhodopsin micelles in phospholipid/detergent mixtures in 150 mM KCl at 22 degrees C, pH 7.3. Neither the replacements by cysteine nor the subsequent labeling affected the absorption spectrum of bacteriorhodopsin and the rise times of the M intermediate. Only the decay of M was altered for some bacteriorhodopsin mutants with cysteine residues on the cytoplasmic side. The proton release time detected with fluorescein attached to the extracellular surface (the proton release side) at position 72 (in the loop connecting helices B and C) or 130 (DE loop) was 22 +/- 4 microseconds, clearly faster than that measured with pyranine in the aqueous bulk phase (125 +/- 10 microseconds for wild-type and all mutants studied). For bacteriorhodopsin mutants labeled at positions 35, 101, 160, 229, and 231 in the cytoplasmic loop region (the proton uptake side), the released proton was observed with a time of 61 +/- 4 microseconds. This was about 3-fold slower than the release time on the extracellular side, but still significantly faster than that measured with pyranine in the bulk phase. These results suggest that the released protons are retained on the micellar surface and move more rapidly along this surface to the cytoplasmic side than from the surface to the bulk medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Coreconstitution of bacterial ATP synthase with monomeric bacteriorhodopsin into liposomes. A comparison between the efficiency of monomeric bacteriorhodopsin and purple membrane patches in coreconstitution experiments.

The conditions for coreconstitution of a bacterial ATP synthase and bacteriorhodopsin into lecithin liposomes and for light driven ATP synthesis have been optimized. A rate of maximally 280 nmol ATP min-1 mg ATP synthase-1 was achieved with monomerized bacteriorhodopsin compared with a rate of up to 45 nmol ATP min-1 mg-1 found for proteoliposomes containing bacteriorhodopsin in the form of purple membrane patches. The different rates are explained by the finding that monomeric bacteriorhodopsin is more homogeneously distributed among the liposomes than the purple membrane patches. The final activities depended on both the purification method for the two proteins and the coreconstitution procedure. Furthermore, the ratio (lipid to bacteriorhodopsin to ATP synthase) could be optimized. Light-driven ATP synthesis depends also on the type of detergent used. The best result was obtained by deoxycholate. Also the relationship between proton translocation (by bacteriorhodopsin) and ATP synthesis activity was measured. A constant H+/ATP ratio was found at higher light intensities. This ratio increased strongly at lower light intensities.

Bacteriorhodopsins↗
PubMed1998

Detection and expression of a gene encoding a new bacteriorhodopsin from an extreme halophile strain HT (JCM 9743) which does not possess bacteriorhodopsin activity.

Membrane vesicles prepared from an extreme halophile strain, HT (JCM 9743), showed no bacteriorhodopsin activity. However, a DNA fragment, amplified by polymerase chain reaction (PCR), appeared to encode the C to G helices of a bacteriorhodopsin (bR)-like protein. With the PCR product as a probe, the gene coding for a novel bacteriorhodopsin was cloned from the genomic DNA of the strain HT. The open reading frame of the gene was ligated with the promoter region of the bop gene of Halobacterium salinarum bR, and expressed in a bR-deficient host strain, L33, using the plasmid vector pXLNov-R. The purplish membrane fraction purified from cells of a transformant exhibited a cyclic photoreaction characteristic of bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1996

Solid-state 13C-NMR of [(3-13C)Pro]bacteriorhodopsin and [(4-13C)Pro]bacteriorhodopsin: evidence for a flexible segment of the C-terminal tail.

The configuration of an Xaa-Pro bond can be determined by solid-state magic-angle-sample-spinning (MASS)-13C-NMR spectroscopy since the chemical shifts of C beta and Cgamma of the proline ring are sensitive to the isomerization state of the preceding peptide bond. (3-13C)Pro and (4-13C)Pro have been chemically synthesized; the former by means of an asymmetric synthesis. The 13C-labeled Pro residues were biosynthetically incorporated into bacteriorhodopsin with a yield of 80%. The solid-state-MASS-13C-NMR spectra of [(3-13C)Pro]bacteriorhodopsin and [(4-13C)Pro]bacteriorhodopsin revealed isotropic chemical shifts at 29.8 ppm and 25.5 ppm, respectively. From the chemical-shift values we conclude that all Xaa Pro peptide bonds are in the trans configuration confirming previous results from solution-NMR studies on solubilized bacteriorhodopsin in organic solvents [Deber, M.C., Sorrell, B.J. & Xu, G.Y. (1990) Biochem. Biophys. Res. Commun. 172, 862-869]. Inversion-recovery experiments could differentiate between three classes of Pro residues distinguished by their relaxation time t1. Tentatively, these three distinct groups of Pro residues could be assigned to the helical, the loop, and the C-terminal parts of the protein. The resonances of the two C-terminal Pro could be identified by removing the C-terminus by proteolysis. Although they are separated by only one Glu they occupy different chemical environments and possess different flexibilities. These results indicate that the first part of the C-terminal tail is constrained. Pro238 marks the position where the tail becomes freely mobile. It is proposed that the C-terminus is fixed to the membrane via salt bridges between divalent cations and negative charges of the C-terminus as well as interhelical loops.

Bacteriorhodopsins↗
PubMed1979

Association of bacteriorhodopsin with lipid-impregnated filters. Evidence for fusion of bacteriorhodopsin-containing vesicles with the lipid phase of the filter.

Bacteriorhodopsin vesicles were associated with cellulose-nitrate filters impregnated with a solution of phospholipids in hexadecane. The generation of (photo)potentials upon illumination of the filter was studied in the absence and presence of ionophores, phospholipase A2, EDTA or polyene antibiotics. From these experiments the following conclusions are drawn. 1. Upon illumination of the filter, bacteriorhodopsin pumps protons into aqueous compartments located in the filter. 2. These aqueous compartments possibly do not originate from the compartments enclosed by the bacteriorhodopsin vesicles. Evidence is obtained that aqueous compartments are present in the surface layers of the lipid-impregnated filters. 3. The results are explained most easily by a mechanism, whereby fusion occurs between the vesicles and the lipids of the filter.

Amphotericin B↗
PubMed1996

ATP synthesis by the F0F1 ATP synthase from thermophilic Bacillus PS3 reconstituted into liposomes with bacteriorhodopsin. 1. Factors defining the optimal reconstitution of ATP synthases with bacteriorhodopsin.

Optimal conditions for the reconstitution of bacteriorhodopsin and H+-transporting ATP synthase from thermophilic Bacillus PS3 (TF0F1) were determined. Phosphatidylcholine/phosphatidic acid liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, octaethylene glycol n-dodecylether, sodium cholate or sodium deoxycholate and the incorporation of proteins by these detergents was studied at each step of the solubilization process. After removal of detergent by means of SM-2 Bio-Beads, the light-driven ATP synthase activities of the resulting proteoliposomes were analyzed at 40 degrees C. The nature of the detergent used for reconstitution was important for determining the mechanism of protein insertions. The most efficient reconstitutions were obtained with octyl glucoside or Triton X-100 by insertion of the proteins into detergent-saturated liposomes. The conditions for reconstitutions were further optimized with regard to functional coupling between bacteriorhodopsin and TF0F1. It was demonstrated that one of the main factors limiting the production of efficient reconstituted proteoliposomes was related to activation of the highly stable TFO-F1. Activation was accomplished by total solubilization of phospholipids and proteins in a Triton X-100/octyl glucoside mixture containing 20 mM octyl glucoside, leading to a threefold stimulation of the ATP synthase activity. Final ATP synthase activities depended greatly on the lipid/bacteriorhodopsin and the lipid/TF0F1 ratios as well as on the phospholipid used. In particular, light-driven ATP synthesis depended upon the presence of negatively charged phospholipids. Cholesterol was found to induce a fourfold increase in ATP synthase activity with a concomitant 65% decrease in the Km for ADP, suggesting that sterols can modulate catalytic events mediated by F1. Preparations obtained by this step-by-step reconstitution procedure displayed activities up to 20-fold higher (500-800 nmol ATP x min(-1) x mg TF0F1(-1) in the presence of cholesterol) than the maximal values reported in the literature for light-driven ATP synthesis TF0F1 measured under similar conditions. This study also allowed rationalization of the different parameters involved in reconstitution experiments and the present simple method is shown to be of general use for preparation of efficient proteoliposomes containing bacteriorhodopsin and choloroplast or mitochondrial F0F1-type ATP synthases.

Adenosine Triphosphate↗
PubMed1991

Structure-function studies of bacteriorhodopsin XV. Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure.

Bacteriorhodopsin mutants containing deletions in loop B-C, delta Thr67-Glu74 or delta Gly65-Gln75 or a deletion in the loop E-F, delta Glu161-Ala168, were prepared. Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4 degrees C but showed the following changes at 20 degrees C. 1) The lambda max shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein.

Amino Acid Sequence↗
PubMed2001

Structure and function in bacteriorhodopsin: the role of the interhelical loops in the folding and stability of bacteriorhodopsin.

Bacteriorhodopsin functions as a light-driven proton pump in Halobacterium salinarium. The functional protein consists of an apoprotein, bacterioopsin, with seven transmembrane alpha helices together with a covalently bound all-trans retinal chromophore. In order to study the role of the interhelical loop conformations in the structure and function of bacteriorhodopsin, we have constructed bacterioopsin genes where each loop is replaced, one at a time, by a peptide linker consisting of Gly-Gly-Ser- repeat sequences, which are believed to have flexible conformations. These mutant proteins have been expressed in Escherichia coli, purified and reconstituted with all-trans retinal in l-alpha-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/3-(3-cholamidopropyl)dimethylammonio-1-propane sulfonate (CHAPS)/SDS and l-alpha-1,2-dihexanoylphosphatidylcholine (DHPC)/DMPC/SDS micelles. Wild-type-like chromophore formation was observed in all the mutants containing single loop replacements. In the BC and FG mutants, an additional chromophore band with an absorption band at about 480 nm was observed, which was in equilibrium with the 550 nm, wild-type band. The position of the equilibrium depended on temperature, SDS and relative DMPC concentration. The proton pumping activity of all of the mutants was comparable to that of wild-type bR except for the BC and FG mutants, which had lower activity. All of the loop mutants were more sensitive to denaturation by SDS than the wild-type protein, except the mutant where the DE loop was replaced. These results suggest that a specific conformation of all the loops of bR, except the DE loop, contributes to bR stability and is required for the correct folding and function of the protein. An increase in the relative proportion of DHPC in DHPC/DMPC micelles, which reduces the micelle rigidity and alters the micelle shape, resulted in lower folding yields of all loop mutants except the BC and DE mutants. This effect of micelle rigidity on the bR folding yield correlated with a loss in stability of a partially folded, seven-transmembrane apoprotein intermediate state in SDS/DMPC/CHAPS micelles. The folding yield and stability of the apoprotein intermediate state both decreased for the loop mutants in the order WT approximately BC approximately DE>FG>AB>EF> or =CD, where the EF and CD loop mutants were the least stable.

Amino Acid Sequence↗
PubMed1999

Formation of the M(N) (M(open)) intermediate in the wild-type bacteriorhodopsin photocycle is accompanied by an absorption spectrum shift to shorter wavelength, like that in the mutant D96N bacteriorhodopsin photocycle.

Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M(N) intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M(N) (M(open)) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 approximately 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (approximately 10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M(N) (M(open)) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.

Bacteriorhodopsins↗
PubMed1989

Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin.

Bacteriorhodopsin contains 8 tryptophan residues distributed across the membrane-embedded helices. To study their possible functions, we have replaced them one at a time by phenylalanine; in addition, Trp-137 and -138 have been replaced by cysteine. The mutants were prepared by cassette mutagenesis of the synthetic bacterio-opsin gene, expression and purification of the mutant apoproteins, renaturation, and chromophore regeneration. The replacement of Trp-10, Trp-12 (helix A), Trp-80 (helix C), and Trp-138 (helix E) by phenylalanine and of Trp-137 and Trp-138 by cysteine did not significantly alter the absorption spectra or affect their proton pumping. However, substitution of the remaining tryptophans by phenylalanine had the following effects. 1) Substitution of Trp-86 (helix C) and Trp-137 gave chromophores blue-shifted by 20 nm and resulted in reduced proton pumping to about 30%. 2) As also reported previously (Hackett, N. R., Stern, L. J., Chao, B. H., Kronis, K. A., and Khorana, H. G. (1987) J. Biol. Chem. 262, 9277-9284), substitution of Trp-182 and Trp-189 (helix F) caused large blue shifts (70 and 40 nm, respectively) in the chromophore and affected proton pumping. 3) The substitution of Trp-86 and Trp-182 by phenylalanine conferred acid instability on these mutants. The spectral shifts indicate that Trp-86, Trp-182, Trp-189, and possibly Trp-137 interact with retinal. It is proposed that these tryptophans, probably along with Tyr-57 (helix B) and Tyr-185 (helix F), form a retinal binding pocket. We discuss the role of tryptophan residues that are conserved in bacteriorhodopsin, halorhodopsin, and the related family of opsin proteins.

Amino Acid Sequence↗
PubMed1996

Catalysis of the retinal subpicosecond photoisomerization process in acid purple bacteriorhodopsin and some bacteriorhodopsin mutants by chloride ions.

The dynamics and the spectra of the excited state of the retinal in bacteriorhodopsin (bR) and its K-intermediate at pH 0 was compared with that of bR and halorhodopsin at pH 6.5. The quantum yield of photoisomerization in acid purple bR was estimated to be at least 0.5. The change of pH from 6.5 to 2 causes a shift of the absorption maximum from 568 to 600 nm (acid blue bR) and decreases the rate of photoisomerization. A further decrease in pH from 2 to 0 shifts the absorption maximum back to 575 nm when HCl is used (acid purple bR). We found that the rate of photoisomerization increases when the pH decreases from 2 to 0. The effect of chloride anions on the dynamics of the retinal photoisomerization of acid bR (pH 2 and 0) and some mutants (D85N, D212N, and R82Q) was also studied. The addition of 1 M HCl (to make acid purple bR, pH 0) or 1 M NaCl to acid blue bR (pH 2) was found to catalyze the rate of the retinal photoisomerization process. Similarly, the addition of 1 M NaCl to the solution of some bR mutants that have a reduced rate of retinal photoisomerization (D85N, D212N, and R82Q) was found to catalyze the rate of their retinal photoisomerization process up to the value observed in wild-type bR. These results are explained by proposing that the bound Cl- compensates for the loss of the negative charges of the COO- groups of Asp85 and/or Asp212 either by neutralization at low pH or by residue replacement in D85N and D212N mutants.

Acoustics↗
PubMed2004

Femtosecond primary events in bacteriorhodopsin and its retinal modified analogs: revision of commonly accepted interpretation of electronic spectra of transient intermediates in the bacteriorhodopsin photocycle.

Femtosecond primary events in bacteriorhodopsin (BR) and its retinal modified analogs are discussed. Ultrafast time resolved electronic spectra of the primary intermediates induced in the BR photocycle are discussed along with spectral and kinetic inconsistencies of the previous models proposed in the literature. The theoretical model proposed in this paper based on vibrational coupling between the electronic transition of the chromophore and intramolecular vibrational modes allows us to calculate the equilibrium electronic absorption band shape and the hole burning profiles. The model is able to rationalize the complex pattern of behavior for the primary events in BR and explain the origin of the apparent inconsistencies between the experiment and the previous theoretical models. The model presented in the paper is based on the anharmonic coupling assumption in the adiabatic approximation using the canonical transformation method for diagonalization of the vibrational Hamiltonian instead of the commonly used perturbation theory. The electronic transition occurs between the Born-Oppenheimer potential energy surfaces with the electron involved in the transition being coupled to the intramolecular vibrational modes of the molecule (chromophore). The relaxation of the excited state occurs by indirect damping (dephasing) mechanisms. The indirect dephasing is governed by the time evolution of the anharmonic coupling constant driven by the resonance energy exchange between the intramolecular vibrational mode and the bath. The coupling with the intramolecular vibrational modes results in the Franck-Condon progression of bands that are broadened due to the vibrational dephasing mechanisms. The electronic absorption line shape has been calculated based on the linear response theory whereas the third order nonlinear response functions have been used to analyze the hole burning profiles obtained from the pump-probe time-resolved measurements. The theoretical treatment proposed in this paper provides a basis for a substantial revision of the commonly accepted interpretation of the primary events in the BR photocycle that exists in the literature.

Bacteriorhodopsins↗
PubMed1992

Evaluation of the predicted secondary structure of bacteriorhodopsin. Prediction of the bovine rhodopsin secondary structure and its sequence similarity with bacteriorhodopsin.

The secondary structure of bacteriorhodopsin (bacR) is predicted using the Chou and Fasman method in conjunction with the hydropathic index of Kyte and Doolittle. The predicted bacR structure was compared with the structure determined by Henderson et al. (1990) using electron diffraction and was found to correlate extremely well. The secondary structure of bovine rhodopsin (bovR) was then predicted using the same techniques. The proposed transmembrane regions of bovR were then examined and found to have sequence similarity with those transmembrane regions of bacR.

Amino Acid Sequence↗
PubMed1994

Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin. 2. Rotational orientation of helices D and E and kinetic correlation between M formation and proton release in bacteriorhodopsin micelles.

The kinetics of the light-induced proton release in bacteriorhodopsin/lipid/detergent micelles was monitored with the optical pH-indicator fluorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton release side). Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sulfhydryl group by reaction with (iodoacetamido)fluorescein. Two characteristic proton release times (approximately 20 and 70 microseconds) were observed. The faster time constant was recorded when fluorescein was attached to positions 127, 130, 131, 132, and 134, while the slower time was observed with the indicator bound to positions 128, 129, and 133. The results are rationalized by assuming specific helical wheel orientations for helics D and E and by making a choice for the residues in the DE loop: (i) The fast time constants occur with fluorescein either attached to residues 130, 131, and 132 that form the DE loop or when pointing toward the interior of the protein with its aqueous proton channel [residues 127 (helix D) and 134 (helix E)]. (ii) The slower time constants are detected with fluorescein exposed to the exterior lipid/detergent phase when bound to residues 128, 129 (both helix D), and 133 (helix E). This interpretation is supported by measurements of the polarity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic environment. The fastest proton release time (10 microseconds) was observed with fluorescein bound to position 127.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗