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D Oesterhelt

Publications and source records attributed to D Oesterhelt.

At least 109 records · Page 6Linked to original sources
PubMed1993

Projection structure of halorhodopsin from Halobacterium halobium at 6 A resolution obtained by electron cryo-microscopy.

Two-dimensional crystals of halorhodopsin (HR), in space group p42(1)2 (a = 102 A) have been obtained using the overexpressing Halobacterium halobium strain D2. An HR membrane fraction with the same buoyant density as purple membrane (HR-PM) was obtained by homogenization and sucrose gradient purification and used for electron cryomicroscopic analysis. Electron micrographs and electron diffraction patterns of HR-PM were recorded at liquid nitrogen temperatures. The micrographs showed significant diffraction out to 9 A resolution optically and to 6 A after computer processing. By combining data from electron micrographs and electron diffraction patterns, a projection map of HR was calculated. The crystal form of the isolated HR consists of one membrane in which alternating halorhodopsin tetramers are oriented in opposite directions across the membrane. It is not known whether this occurs by misinsertion of some of the molecules in vivo, or by adventitious fusion at some point during isolation. The projected structure of the HR molecule to a resolution of 6A is almost identical to that found for bacteriorhodopsin (BR). This physical structural similarity thus complements the known sequence relatedness to BR.

Bacteriorhodopsins↗
PubMed1993

Orthorhombic crystal form of bacteriorhodopsin nucleated on benzamidine diffracting to 3.6 A resolution.

Freshly formed benzamidine crystals were found to provide a suitable nucleation surface for crystallisation of bacteriorhodopsin. At 20 degrees C and 1% octylglucoside pseudohexagonal needles of bacteriorhodopsin nucleated on the benzamidine surface. At 4 degrees C and reduced detergent concentration (0.5%) a new, better-ordered orthorhombic crystal form of bacteriorhodopsin was formed by heterogeneous nucleation on benzamidine. Polarised absorption spectroscopy and flash photolysis experiments were used to show that the crystalline bacteriorhodopsin is photoactive in both forms. The M-intermediate absorbs maximally at 405nm and formation of M does not disturb the crystal lattice. The plate-shaped crystals diffract to a resolution of 3.6 A along the a and b directions and to 4.2 A in the c direction. The most likely space group of the crystals is C222 (a = 107.5 A, b = 117.0 A, c = 69.5 A). The crystals are built from layers of bacteriorhodopsin molecules that are tilted from the c axis by about 45 degrees. In addition, as a background to the discovery of the new crystal form, the influence of different detergents, additives and precipitants on the formation of pseudohexagonal needles is presented.

Bacteriorhodopsins↗
PubMed1993

Bacteriorhodopsin is involved in halobacterial photoreception.

The bacterio-opsin gene was introduced into a "blind" Halobacterium salinarium mutant that (i) lacked all the four retinal proteins [bacteriorhodopsin (BR), halorhodopsin, and sensory rhodopsins (SRs) I and II] and the transducer protein for SRI and (ii) showed neither attractant response to long wavelength light nor repellent response to short wavelength light. The resulting transformed cells acquired the capability to sense light stimuli. The cells accumulated in a light spot, demonstrating the BR-mediated orientation in spatial light gradients. As in wild-type cells, a decrease in the intensity of long wavelength light caused a repellent response by inducing reversals of swimming direction, but, in contrast to wild-type cells, a decrease in the intensity of short wavelength light also repelled the cells. An increase in light intensity evoked an attractant response (i.e., a transient suppression of spontaneous reversals). Signal processing times and adaptation kinetics were similar to the SRI-mediated reactions. However, compared to SR-mediated photoresponses, higher light intensities were necessary to induce the BR-mediated responses. The light sensitivity of the transformant was increased by adding 1 mM cyanide and decreased by the addition of arginine, agents that respectively reduce and increase the light-independent generation of the electrochemical potential difference of H+ ions (delta mu H+). A decrease in irradiance to an intensity that was still high enough to saturate BR-initiated delta mu H+ changes failed to induce the repellent effect, but the addition of a protonophorous uncoupler sensitized the cell to these light stimuli. The BR D96N mutant (Asp-96 is replaced by Asn) with decreased proton pump activity showed strongly reduced BR-mediated responses. Azide, which increases this mutant's H+ pump efficiency, increased the photosensitivity of the mutant cells. Moreover, azide diminished (i) the membrane potential decreasing and (ii) repellent effects of blue light added to the orange background illumination in this mutant. We conclude that the BR-mediated photoreception is due to the light-dependent generation of delta mu H+. Our data are consistent with the assumption that the H. salinarium cell monitors the membrane energization level with a "protometer" system measuring total delta mu H+ changes or its electric potential difference component.

Bacteriorhodopsins↗
PubMed1993

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 1. Site-directed mutagenesis and initial characterization.

Five site-directed mutants were engineered to substitute phenylalanine, serine, leucine, methionine, and glycine for tyrosine residue 162 of the pufL gene in Rhodobacter (R.) sphaeroides. Each of the mutations and the wild-type (WT) genes was expressed in the R. sphaeroides puf deletion strain PUF delta LMX21/3. Initial characterization revealed that all of the mutants were photoheterotrophically competent but that L162G and L162S were impaired. The amounts of mutant reaction centers expressed, the spectral characteristics, and the rates of intraprotein electron transfer and turnover were similar to the values obtained for WT. Kinetic measurements of photooxidized special pair rereduction mediated by the physiological donor cytochrome c2 in intact chemoheterotrophically grown cells revealed that the fast phase was abolished in all mutants and that the overall kinetics of rereduction was drastically slowed. It is concluded that L162Y plays a vital role in facilitating the rapid rereduction of the photooxidized bacteriochlorophyll dimer in R. sphaeroides.

Amino Acid Sequence↗
PubMed1993

Tyrosine 162 of the photosynthetic reaction center L-subunit plays a critical role in the cytochrome c2 mediated rereduction of the photooxidized bacteriochlorophyll dimer in Rhodobacter sphaeroides. 2. Quantitative kinetic analysis.

The electron-transfer kinetics from the soluble cytochrome (cyt) c2 to the photooxidized reaction center (RC) was studied with proteins isolated from Rhodobacter (R.) sphaeroides. In addition to wild-type (WT) RC, RCs harboring site-directed mutations at residue L162 (L162F, -M, -L, -S, or -G) wree analyzed. The disappearance of the absorption band of the photooxidized primary donor P+ (at 1250 nm) and the alpha-band of cyt c2 (at 550 nm) were monitored. Under conditions of high equimolar RC and cyt c2 concentrations, the kinetics were very similar to those measured in intact cells (Farchaus et al., 1993). The fast component of the kinetics normally seen in WT was not observed in any of the mutants; the overall rereduction rates for the mutants depended on the amino acid substitution. Light intensity, viscosity, ionic strength, and RC/cyt c2 stoichiometry of the reaction mixture were varied to distinguish the contributions of association, reorientation, and electron-transfer reactions to the observed kinetics. In competition experiments, WTRC (L162Y) and the mutant RCL162L showed similar affinity for cyt c2, with a dissociation constant of kD = 10(-6) M. Mutants with an aliphatic substitution at position L162 displayed slower cyt c2-RC association and dissociation rates. Comparison of the major kinetic component of the P+ rereduction rates for the aliphatic substitutions to the aromatic substitution, L162F, revealed that the former were less affected by ionic strength and viscosity than the latter. The viscosity and ionic strength dependences noted for L162F were comparable to those seen for the slow kinetic component observed for the WT RC. The redox midpoint potential of the P/P+ couple was increased by 30 mV (L162F) to 50 mV (L162L, G) over the WT value, leading to differences in delta G not large enough to account for the drastic kinetic effects. Rather, the results suggested that the state(s) where cyt c2 is nonproductively bound to the RC dominated in the mutants. In the L162F mutant, it appeared that only the distribution between the bound cyt c2 states was affected, whereas for the mutants with aliphatic substitutions, a decreased reorientation rate had to be additionally assumed in order to explain the observations.

Amino Acid Sequence↗
PubMed1993

Homologous bacterio-opsin-encoding gene expression via site-specific vector integration.

Homologous recombination in the archaebacterium Halobacterium halobium has been investigated and exploited for the wild-type (wt) level of expression of the bacterio-opsin-encoding gene (bop). The Haloferax volcanii-Escherichia coli shuttle vector, pWL102, was used to construct a shuttle-mutagenesis vector, pEF191, bearing bop and short flanking sequences. Transformation of a bacteriorhodopsin (BR)-negative H. halobium strain with pEF191 resulted in plasmid integration at the homologous bop locus. A model for this site-specific vector integration is presented which has been confirmed by determining the arrangement of the repeated homologous sequences on the chromosome. Two different configurations are obtained after integrative transformation due to the presence of an insertion element in the genomic copy of bop. In one configuration, the functional bop cluster containing the regulatory bat and brp genes was in wt arrangement. In the second configuration, the bop cluster is interrupted by 10 kb of plasmid vector sequences, and the upstream region required for bop expression was limited to 400 bp. The BR production for both configurations was determined and found to be at wt level. These results suggest that the function of the putative bop promoter does not depend on the defined upstream positions of bat and brp. The system presented here can be easily exploited for structure-function studies on BR and introduces homologous gene targeting as a powerful tool in the study of halobacterial genetics.

Bacteriorhodopsins↗
PubMed1993

Light-driven proton or chloride pumping by halorhodopsin.

Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base.

Azides↗
PubMed1993

Electron diffraction analysis of structural changes in the photocycle of bacteriorhodopsin.

Structural changes are central to the mechanism of light-driven proton transport by bacteriorhodopsin, a seven-helix membrane protein. The main intermediate formed upon light absorption is M, which occurs between the proton release and uptake steps of the photocycle. To investigate the structure of the M intermediate, we have carried out electron diffraction studies with two-dimensional crystals of wild-type bacteriorhodopsin and the Asp96-->Gly mutant. The M intermediate was trapped by rapidly freezing the crystals in liquid ethane following illumination with a xenon flash lamp at 5 and 25 degrees C. Here, we present 3.5 A resolution Fourier projection maps of the differences between the M intermediate and the ground state of bacteriorhodopsin. The most prominent structural changes are observed in the vicinity of helices F and G and are localized to the cytoplasmic half of the membrane.

Amino Acid Sequence↗
PubMed1993

The methyl-accepting transducer protein HtrI is functionally associated with the photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium.

We have investigated the functional relationship between two proteins involved in the photosensory system of the archaeon Halobacterium salinarium: the photoreceptor sensory rhodopsin I (SRI) and the halobacterial transducer rhodopsin I (HtrI), which has been proposed to be the putative signal transducer of SRI, by genomic DNA analysis of two independent SRI negative mutants, Pho81 and D1. Southern and PCR analyses revealed that both strains bear alterations in the 5' flanking region of the gene encoding SRI, sopI. DNA sequence analysis confirmed the occurrence in this region of htrI, the gene encoding the putative transducer protein. PCR and Northern analyses have shown further that sopI and htrI are expressed as a single transcriptional unit, thus explaining the lack of SRI in mutants with a defective htrI. Expression of the cloned sopI under the control of a heterologous promoter did not restore the SRI-dependent photoresponse in the strain Pho81. Moreover, the photocycling rate of the expressed pigment was clearly lower than in wild type. HtrI is therefore essential for SRI function and most likely modulates the photochemical properties of the photoreceptor via direct physical interaction. Finally, reintroduction of both sopI and htrI into Pho81 and D1 restored the SRI photochemistry and its physiological function. Our results provide the first experimental evidence for the functional coupling between SRI and HtrI and corroborate the proposed model in which HtrI acts as the signal transducer of this archaeal seven-helix photoreceptor in a way analogous to the bacterial chemotaxis transducers.

Bacterial Proteins↗
PubMed1993

Decoupling of photo- and proton cycle in the Asp85-->Glu mutant of bacteriorhodopsin.

Surface bound pH indicators were applied to study the proton transfer reactions in the mutant Asp85-->Glu of bacteriorhodopsin in the native membrane. The amino acid replacement induces a drastic acceleration of the overall rise of the M intermediate. Instead of following this acceleration, proton ejection to the extracellular membrane surface is not only two orders of magnitude slower than M formation, it is also delayed as compared with the wild-type. This demonstrates that Asp85 not only accepts the proton released by the Schiff's base but also regulates very efficiently proton transfer within the proton release chain. Furthermore, Asp85 might be the primary but is not the only proton acceptor/donor group in the release pathway. The Asp85-->Glu substitution also affects the proton reuptake reaction at the cytoplasmic side, although Asp85 is located in the proton release pathway. Proton uptake is slower in the mutant than in the wild-type and occurs during the lifetime of the O intermediate. This demonstrates a feed-back mechanism between Asp85 and the proton uptake pathway in bacteriorhodopsin.

Asparagine↗
PubMed1993

The fdx gene encoding the [2Fe--2S] ferredoxin of Halobacterium salinarium (H. halobium).

The gene encoding the [2Fe--2S] ferredoxin (fdx gene) was isolated from Halobacterium salinarium using two oligonucleotides deduced from the ferredoxin sequence as probes. Cosmid DNAs exhibiting hybridization were isolated, the fdx gene was localized to smaller subfragments and the nucleotide sequence determined. The 390 bp coding sequence is located in the halobacterial FI-DNA and transcribed as a 440 nucleotide mRNA. S1 mapping indicated that the 5' terminus of the mRNA maps immediately upstream of the ATG start codon. The promoter box A, centred around position -25 (5' AC-TATG 3'), and box B (TG) elements at the start of the transcript resemble the sequences of a typical archaeal promoter. The restriction pattern of an approximately 50 kb region surrounding the fdx gene is conserved in various Halobacterium species. The halobacterial ferredoxin and the major gas vesicle protein GvpA exhibit up to 70% similarity to their respective counterparts in cyanobacteria suggesting lateral gene transfer between the organisms. These similarities prompted a more detailed investigation of the relative positions of the genes in the halobacterial genome.

Amino Acid Sequence↗
PubMed1993

Photochemical conversion of the O-intermediate to 9-cis-retinal-containing products in bacteriorhodopsin films.

The photochemical activity of the O-state was investigated in bacteriorhodopsin (BR) films containing wildtype BR at pH 6.5 in the presence of glycerol. The formation of a photoproduct of O with an absorption maximum at 490 nm and 9-cis-retinal configuration was found. This 490-nm product was named P and shows a slow thermal reaction into a compound with a maximal absorption at 380 nm which was named Q and contains free 9-cis-retinal in the proteins binding site. The photoproducts of O, i.e., P and Q, are very similar, or even identical, to those previously observed in blue membranes. Common to the O-state and blue membrane forms of bacteriorhodopsin is a protonated aspartic acid 85, and we suggest that it is the reduced negative charge around the Schiff base which is responsible for the 9-cis photoisomerization. The release of a proton from aspartic acid 85 is linked to the conversion of the O-state back to the initial state of BR. Therefore the conditions of low proton mobility in BR films containing glycerol favor the accumulation of the O-state. For optical and holographic applications such BR films are very attractive. It is possible to create photoproducts with red light which are thermally stable at room temperature and that can be photochemically erased. Dependent on the light composition both properties can be realized in the same sample material. This feature may bridge the gap between information processing and short-term and long-term storage of information with BR.

Bacteriorhodopsins↗
PubMed1993

Sensory rhodopsin-controlled release of the switch factor fumarate in Halobacterium salinarium.

Halobacterium salinarium responds to blue light by reversing its swimming direction. Fumarate has been proposed as one of the molecular components of this sensory system and is involved in the switching process of the flagellar motor. In order to obtain chemical proof for this role of fumarate, cells were stimulated with a pulse of blue light and lysed by rapid mixing with distilled water. The lysate contained fumarate in free and bound form, which were separated by ultrafiltration. The fumarate concentration in the low-molecular-mass fraction (< 5 kDa) of the lysate was assayed enzymatically and a light-induced increase was observed. Additionally, the total cellular fumarate content decreased in response to light, indicating that fumarate was released from a cellular pool rather than being formed by de novo synthesis. The light-induced release was not detected in a mutant defective in sensory rhodopsin-I and -II. Therefore it is concluded that photoreceptor activation rather than a direct effect of light on the activity of metabolic enzymes causes fumarate release. For each photoactivated sensory rhodopsin-II molecule at least 350 molecules of fumarate were liberated demonstrating efficient amplification. The rate of light-induced fumarate release is at least 10-times faster than the fumarate turnover number of the citric acid cycle which was estimated as approximately 4300 per cell and second. Therefore this metabolic process is not expected to be part of the signal transduction chain in the halobacterial cell.

Cell Membrane↗
PubMed1993

Homologous overexpression of a light-driven anion pump in an archaebacterium.

The retinal protein halorhodopsin (HR), a light-driven chloride pump from Halobacterium halobium, was homologously overexpressed in this archaebacterium. Two DNA expression systems differing in their promoter region were investigated. The halopsin, hop, promoter coupled to the hop gene gave an increased level of HR synthesis. However, the extent of expression was driven by the copy number of the shuttle vector and did not reach the magnitude of the bacterio-opsin, bop, promoter system. Employing a gene fusion approach, the promoter for the bop gene was used to drive expression of the hop gene. A shuttle vector containing a bop-hop-cartridge was transformed into a HR-deficient strain and blueish-coloured transformants were obtained. The bop promoter expressed HR to an extent where a specific membrane fraction resembled the crystalline purple membrane of BR in terms of the lipid to protein ratio. HR could, therefore, be easily isolated in a natural membrane-bound state. This allows for direct use in biophysical studies without the application of detergents. This was the first successful overexpression of a 7-helical transmembrane protein and may be extended to other proteins of this family.

Amino Acid Sequence↗
PubMed1993

A C-terminal truncation results in high-level expression of the functional photoreceptor sensory rhodopsin I in the archaeon Halobacterium salinarium.

Expression of the gene encoding the halobacterial photoreceptor sensory rhodopsin I (SRI), sopI, was studied by means of homologous gene targeting. A sopI- Halobacterium salinarium mutant strain was constructed by homologous replacement of sopI with a novobiocin-resistant gyrB from Haloferax Aa 2.2. Cells bearing gyrB were resistant to novobiocin, indicating that the Haloferax gene is functional in H. salinarium. Complementation of this deletion strain with sopI fused to the bacterio-opsin promoter resulted in the recovery of all phenotypical attributes of SRI. This establishes the first direct correlation between sopI and the function of its gene product. In the complemented deletion strain, functional expression of sopI occurred from the bop locus, where sopI had integrated by homologous recombination. This shows that cotranscription of sopI and the gene encoding the SRI signal transducer, htrI, which is found in the wild type, is not a prerequisite for photosensory activity. Deletion of the last 43 bp at the 3' end of sopI resulted in a 10-fold increase in the amount of SRI, without affecting the activity of the pigment. The mRNA level of the truncated gene was not affected as compared to that of the wild type. We propose that regulation occurs at the protein level, probably through a negative determinant of protein stability located in the C-terminus of SRI. Replacement of the last 28 amino acids of bacteriorhodopsin by the last 29 amino acids of SRI results in a decrease of the bacteriorhodopsin, supporting our observations. The C-terminus of SRI is the first domain with a downregulating influence on protein levels thus far identified in H. salinarium. The system for SRI overexpression we present here greatly facilitates biochemical and biophysical studies on the photoreceptor and allows investigation of the molecular interactions underlying the signal transduction chain of halobacterial phototaxis.

Amino Acid Sequence↗
PubMed1993

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.

Amino Acid Sequence↗