536 research outputs found
Archaebacterial rhodopsin sequences: Implications for evolution
It was proposed over 10 years ago that the archaebacteria represent a separate kingdom which diverged very early from the eubacteria and eukaryotes. It follows that investigations of archaebacterial characteristics might reveal features of early evolution. So far, two genes, one for bacteriorhodopsin and another for halorhodopsin, both from Halobacterium halobium, have been sequenced. We cloned and sequenced the gene coding for the polypeptide of another one of these rhodopsins, a halorhodopsin in Natronobacterium pharaonis. Peptide sequencing of cyanogen bromide fragments, and immuno-reactions of the protein and synthetic peptides derived from the C-terminal gene sequence, confirmed that the open reading frame was the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences of this gene, as well as those of other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences were calculated. These indicate very considerable evolutionary distance between each pair of genes, even in the dame organism. In spite of this, three protein sequences show extensive similarities, indicating strong selective pressures
Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR
Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 ± 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F- , citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride- transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site.
We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites
High-precision Measurements of Ionospheric TEC Gradients with the Very Large Array VHF System
We have used a relatively long, contiguous VHF observation of a bright cosmic
radio source (Cygnus A) with the Very Large Array (VLA) to demonstrate the
capability of this instrument to study the ionosphere. This interferometer, and
others like it, can observe ionospheric total electron content (TEC)
fluctuations on a much wider range of scales than is possible with many other
instruments. We have shown that with a bright source, the VLA can measure
differential TEC values between pairs of antennas (delta-TEC) with an precision
of 0.0003 TECU. Here, we detail the data reduction and processing techniques
used to achieve this level of precision. In addition, we demonstrate techniques
for exploiting these high-precision delta-TEC measurements to compute the TEC
gradient observed by the array as well as small-scale fluctuations within the
TEC gradient surface. A companion paper details specialized spectral analysis
techniques used to characterize the properties of wave-like fluctuations within
this data.Comment: accepted for publication in Radio Scienc
Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses
Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes
Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes†
ABSTRACT: Attractant and repellent signaling conformers of the dual-signaling phototaxis receptor sensory rhodopsin I and its transducer subunit (SRI-HtrI) have recently been distinguished experimentally by the opposite connection of their retinylidene protonated Schiff bases to the outwardly located periplasmic side and inwardly located cytoplasmic side. Here we show that the pKa of the outwardly located Asp76 counterion in the outwardly connected conformer is lowered by ∼1.5 units from that of the inwardly connected conformer. The pK a difference enables quantitative determination of the relative amounts of the two conformers in wild-type cells and behavioral mutants prior to photoexcitation, comparison of their absorption spectra, and determination of their relative signaling efficiency. We have shown that the onephoton excitation of the SRI-HtrI attractant conformer causes a Schiff base connectivity switch from inwardly connected to outwardly connected states in the attractant signaling photoreaction. Conversely, a second near-UV photon drives the complex back to the inwardly connected conformer in the repellent signaling photoreaction. The results suggest a model of the color-discriminating dual-signaling mechanism in which phototaxis responses (his-kinase modulation) result from the photointerconversion of the two oppositely connected SRI-HtrI conformers by one-photon and two-photon activation. Furthermore, we find that the related repellent phototaxis SRII-HtrII receptor complex has an outwardly connecte
Active Membrane Fluctuations Studied by Micropipet Aspiration
We present a detailed analysis of the micropipet experiments recently
reported in J-B. Manneville et al., Phys. Rev. Lett. 82, 4356--4359 (1999),
including a derivation of the expected behaviour of the membrane tension as a
function of the areal strain in the case of an active membrane, i.e.,
containing a nonequilibrium noise source. We give a general expression, which
takes into account the effect of active centers both directly on the membrane,
and on the embedding fluid dynamics, keeping track of the coupling between the
density of active centers and the membrane curvature. The data of the
micropipet experiments are well reproduced by the new expressions. In
particular, we show that a natural choice of the parameters quantifying the
strength of the active noise explains both the large amplitude of the observed
effects and its remarkable insensitivity to the active-center density in the
investigated range. [Submitted to Phys Rev E, 22 March 2001]Comment: 14 pages, 5 encapsulated Postscript figure
Structural Basis for the Aminoacid Composition of Proteins from Halophilic Archea
In order to survive in highly saline environments, proteins from halophilic archea have evolved with biased amino acid compositions that have the capacity to reduce contacts with the solvent
Removal and Reconstitution of the Carotenoid Antenna of Xanthorhodopsin
Salinixanthin, a C40-carotenoid acyl glycoside, serves as a light-harvesting antenna in the retinal-based proton pump xanthorhodopsin of Salinibacter ruber. In the crystallographic structure of this protein, the conjugated chain of salinixanthin is located at the protein–lipid boundary and interacts with residues of helices E and F. Its ring, with a 4-keto group, is rotated relative to the plane of the π-system of the carotenoid polyene chain and immobilized in a binding site near the β-ionone retinal ring. We show here that the carotenoid can be removed by oxidation with ammonium persulfate, with little effect on the other chromophore, retinal. The characteristic CD bands attributed to bound salinixanthin are now absent. The kinetics of the photocycle is only slightly perturbed, showing a 1.5-fold decrease in the overall turnover rate. The carotenoid-free protein can be reconstituted with salinixanthin extracted from the cell membrane of S. ruber. Reconstitution is accompanied by restoration of the characteristic vibronic structure of the absorption spectrum of the antenna carotenoid, its chirality, and the excited-state energy transfer to the retinal. Minor modification of salinixanthin, by reducing the carbonyl C=O double bond in the ring to a C-OH, suppresses its binding to the protein and eliminates the antenna function. This indicates that the presence of the 4-keto group is critical for carotenoid binding and efficient energy transfer
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