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Circular dichroism and cross-linking studies of bacteriorhodopsin mutants.
Karnaukhova E, Schey KL, Crouch RK
(2006) Amino Acids 30: 17-23
MeSH Terms: Amino Acid Substitution, Bacteriorhodopsins, Circular Dichroism, Cross-Linking Reagents, Halobacterium salinarum, Mutagenesis, Site-Directed, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Show Abstract · Added May 27, 2014
Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.
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Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple membranes containing 15N-labeled arginine.
Xiao Y, Hutson MS, Belenky M, Herzfeld J, Braiman MS
(2004) Biochemistry 43: 12809-18
MeSH Terms: Arginine, Bacteriorhodopsins, Hydrogen-Ion Concentration, Kinetics, Nitrogen Isotopes, Photochemistry, Protons, Purple Membrane, Spectroscopy, Fourier Transform Infrared, Temperature, Time Factors
Show Abstract · Added May 20, 2014
Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), because its mutation usually results in loss of fast H(+) release, an important step in the normal light-induced H(+) transport mechanism. To help to clarify the structural changes in Arg-82 associated with the H(+)-release step, we have measured time-resolved FT-IR difference spectra of wild-type bR containing either natural-abundance isotopes ((14)N-Arg-bR) or all seven arginines selectively and uniformly labeled with (15)N at the two eta-nitrogens ((15)N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm(-1) vibrational difference band due to the M photocycle intermediate of (14)N-Arg-bR loses substantial intensity in (15)N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is only observed at pH 7 and not at pH 4 where fast H(+) release is blocked. These observations support the earlier conclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibration of Arg-82 can be assigned to a highly perturbed frequency near 1555 cm(-1) in the M state of wild-type bR [Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compound spectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent with a nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.
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Structural and functional characterization of pi bulges and other short intrahelical deformations.
Cartailler JP, Luecke H
(2004) Structure 12: 133-44
MeSH Terms: Amino Acid Motifs, Amino Acid Sequence, Antigens, CD1, Bacteriorhodopsins, Databases, Protein, Hydrogen Bonding, Models, Molecular, Molecular Sequence Data, Phosphatidylinositols, Protein Conformation, Stress, Mechanical
Show Abstract · Added April 15, 2010
We data-mined the Protein Data Bank for short intrahelical deformations, including pi bulges. These are defined as a contiguous stretch of intrahelical residues deviating from the standard alpha-helical i-->i-4 hydrogen bonding pattern, bilaterally flanked by at least one alpha-helical turn resulting in a helix kink of less than 40 degrees. We find that such motifs exist in 4.7% of a PDB subset filtered by quality metrics (resolution <2.5 A, R-factor <0.25, sequence identity <35%). These are typically characterized by at least one i-->i-5 main chain hydrogen bond, with energetically favorable main chain dihedral angles, followed by a variable number of main chain carbonyl groups that do not accept intrahelical main chain hydrogen bonds. Their stabilization commonly occurs via hydrogen bonding to water molecules or polar groups. Numerous deformations are implicated in basic yet vital functional roles, commonly as ligand binding site contributors.
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11 MeSH Terms
X-ray crystallographic analysis of lipid-protein interactions in the bacteriorhodopsin purple membrane.
Cartailler JP, Luecke H
(2003) Annu Rev Biophys Biomol Struct 32: 285-310
MeSH Terms: Bacteriorhodopsins, Binding Sites, Crystallography, X-Ray, Macromolecular Substances, Membrane Lipids, Models, Molecular, Molecular Conformation, Motion, Protein Binding, Protein Conformation, Purple Membrane
Show Abstract · Added April 15, 2010
The past decade has witnessed increasingly detailed insights into the structural mechanism of the bacteriorhodopsin photocycle. Concurrently, there has been much progress within our knowledge pertaining to the lipids of the purple membrane, including the discovery of new lipids and the overall effort to localize and identify each lipid within the purple membrane. Therefore, there is a need to classify this information to generalize the findings. We discuss the properties and roles of haloarchaeal lipids and present the structural data as individual case studies. Lipid-protein interactions are discussed in the context of structure-function relationships. A brief discussion of the possibility that bacteriorhodopsin functions as a light-driven inward hydroxide pump rather than an outward proton pump is also presented.
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Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate.
Rouhani S, Cartailler JP, Facciotti MT, Walian P, Needleman R, Lanyi JK, Glaeser RM, Luecke H
(2001) J Mol Biol 313: 615-28
MeSH Terms: Amino Acid Substitution, Bacterial Proteins, Bacteriorhodopsins, Binding Sites, Crystallography, X-Ray, Cytoplasm, Halobacterium, Hydrogen Bonding, Isomerism, Models, Molecular, Protein Structure, Secondary, Retinaldehyde, Schiff Bases
Show Abstract · Added April 15, 2010
Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.
Copyright 2001 Academic Press.
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13 MeSH Terms
Halide dependence of the halorhodopsin photocycle as measured by time-resolved infrared spectra.
Hutson MS, Shilov SV, Krebs R, Braiman MS
(2001) Biophys J 80: 1452-65
MeSH Terms: Bacteriorhodopsins, Bromides, Halobacterium salinarum, Halorhodopsins, Kinetics, Light, Potassium Chloride, Potassium Compounds, Retinaldehyde, Spectroscopy, Fourier Transform Infrared, Thermodynamics, Time Factors
Show Abstract · Added May 20, 2014
Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(2) describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the tau(1) decay, in a state previously defined as hK(L) (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(1) is probably best described as an hK(L)/hL(1) equilibrium, instead of a single hK(L) state. Different halides affect the relative amounts of hK(L) and hL(1) present, i.e., Cl- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.
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12 MeSH Terms
Evidence for a perturbation of arginine-82 in the bacteriorhodopsin photocycle from time-resolved infrared spectra.
Hutson MS, Alexiev U, Shilov SV, Wise KJ, Braiman MS
(2000) Biochemistry 39: 13189-200
MeSH Terms: Alanine, Arginine, Bacteriorhodopsins, Carboxylic Acids, Halobacterium salinarum, Mutagenesis, Site-Directed, Photochemistry, Protons, Spectroscopy, Fourier Transform Infrared, Temperature
Show Abstract · Added May 20, 2014
Arginine-82 (R82) of bacteriorhodopsin (bR) has long been recognized as an important residue due to its absolute conservation in the archaeal rhodopsins and the effects of R82 mutations on the photocycle and proton release. However, the nature of interactions between R82 and other residues of the protein has remained difficult to decipher. Recent NMR studies showed that the two terminal nitrogens of R82 experience a highly perturbed asymmetric environment during the M state trapped at cryogenic temperatures [Petkova et al. (1999) Biochemistry 38, 1562-1572]. Although previous low-temperature FT-IR spectra of wild-type and mutant bR samples have demonstrated effects of R82 on vibrations of other amino acid side chains, no bands in these spectra were assignable to vibrations of R82 itself. We have now measured time-resolved FT-IR difference spectra of bR intermediates in the wild-type and R82A proteins, as well as in samples of the R82C mutant with and without thioethylguanidinium attached via a disulfide linkage at the unique cysteine site. Several bands in the bR --> M difference spectrum are attributable to guanidino group vibrations of R82, based on their shift upon isotope substitution of the thioethylguanidinium attached to R82C and on their disappearance in the R82A spectrum. The frequencies and intensities of these IR bands support the NMR-based conclusion that there is a significant perturbation of R82 during the bR photocycle. However, the unusually low frequencies attributable to R82 guandino group vibrations in M, approximately 1640 and approximately 1545 cm(-)(1), would require a reexamination of a previously discarded hypothesis, namely, that the perturbation of R82 involves a change in its ionization state.
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10 MeSH Terms
Coupling photoisomerization of retinal to directional transport in bacteriorhodopsin.
Luecke H, Schobert B, Cartailler JP, Richter HT, Rosengarth A, Needleman R, Lanyi JK
(2000) J Mol Biol 300: 1237-55
MeSH Terms: Amino Acid Substitution, Bacteriorhodopsins, Crystallography, X-Ray, Cytoplasm, Hydrogen Bonding, Ion Transport, Isomerism, Light, Membrane Proteins, Models, Molecular, Molecular Sequence Data, Mutation, Protein Structure, Secondary, Protons, Retinaldehyde, Schiff Bases, Static Electricity, Structure-Activity Relationship, Water
Show Abstract · Added April 15, 2010
In order to understand how isomerization of the retinal drives unidirectional transmembrane ion transport in bacteriorhodopsin, we determined the atomic structures of the BR state and M photointermediate of the E204Q mutant, to 1.7 and 1.8 A resolution, respectively. Comparison of this M, in which proton release to the extracellular surface is blocked, with the previously determined M in the D96N mutant indicates that the changes in the extracellular region are initiated by changes in the electrostatic interactions of the retinal Schiff base with Asp85 and Asp212, but those on the cytoplasmic side originate from steric conflict of the 13-methyl retinal group with Trp182 and distortion of the pi-bulge of helix G. The structural changes suggest that protonation of Asp85 initiates a cascade of atomic displacements in the extracellular region that cause release of a proton to the surface. The progressive relaxation of the strained 13-cis retinal chain with deprotonated Schiff base, in turn, initiates atomic displacements in the cytoplasmic region that cause the intercalation of a hydrogen-bonded water molecule between Thr46 and Asp96. This accounts for the lowering of the pK(a) of Asp96, which then reprotonates the Schiff base via a newly formed chain of water molecules that is extending toward the Schiff base.
Copyright 2000 Academic Press.
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19 MeSH Terms
Structural changes in bacteriorhodopsin during ion transport at 2 angstrom resolution.
Luecke H, Schobert B, Richter HT, Cartailler JP, Lanyi JK
(1999) Science 286: 255-61
MeSH Terms: Bacteriorhodopsins, Binding Sites, Crystallography, X-Ray, Cytoplasm, Hydrogen Bonding, Hydrogen-Ion Concentration, Ion Transport, Isomerism, Light, Models, Molecular, Photolysis, Photons, Point Mutation, Protein Conformation, Protein Structure, Secondary, Proton Pumps, Protons, Retinaldehyde, Schiff Bases, Thermodynamics, Water
Show Abstract · Added May 7, 2010
Crystal structures of the Asp96 to Asn mutant of the light-driven proton pump bacteriorhodopsin and its M photointermediate produced by illumination at ambient temperature have been determined to 1.8 and 2.0 angstroms resolution, respectively. The trapped photoproduct corresponds to the late M state in the transport cycle-that is, after proton transfer to Asp85 and release of a proton to the extracellular membrane surface, but before reprotonation of the deprotonated retinal Schiff base. Its density map describes displacements of side chains near the retinal induced by its photoisomerization to 13-cis,15-anti and an extensive rearrangement of the three-dimensional network of hydrogen-bonded residues and bound water that accounts for the changed pKa values (where Ka is the acid constant) of the Schiff base and Asp85. The structural changes detected suggest the means for conserving energy at the active site and for ensuring the directionality of proton translocation.
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21 MeSH Terms
Structure of bacteriorhodopsin at 1.55 A resolution.
Luecke H, Schobert B, Richter HT, Cartailler JP, Lanyi JK
(1999) J Mol Biol 291: 899-911
MeSH Terms: Bacteriorhodopsins, Binding Sites, Crystallography, X-Ray, Hydrogen Bonding, Lipids, Models, Molecular, Protein Conformation, Protein Structure, Secondary, Retinaldehyde, Schiff Bases, Static Electricity
Show Abstract · Added May 7, 2010
Th?e atomic structure of the light-driven ion pump bacteriorhodopsin and the surrounding lipid matrix was determined by X-ray diffraction of crystals grown in cubic lipid phase. In the extracellular region, an extensive three-dimensional hydrogen-bonded network of protein residues and seven water molecules leads from the buried retinal Schiff base and the proton acceptor Asp85 to the membrane surface. Near Lys216 where the retinal binds, transmembrane helix G contains a pi-bulge that causes a non-proline? kink. The bulge is stabilized by hydrogen-bonding of the main-chain carbonyl groups of Ala215 and Lys216 with two buried water molecules located between the Schiff base and the proton donor Asp96 in the cytoplasmic region. The results indicate extensive involvement of bound water molecules in both the structure and the function of this seven-helical membrane protein. A bilayer of 18 tightly bound lipid chains forms an annulus around the protein in the crystal. Contacts between the trimers in the membrane plane are mediated almost exclusively by lipids.
Copyright 1999 Academic Press.
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11 MeSH Terms