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B Roux

Publications and source records attributed to B Roux.

At least 55 records · Page 3Linked to original sources
PubMed1997

Recombinant plasmid mobilization between E. coli strains in seven sterile microcosms.

Transfer by mobilization of a pBR derivative recombinant plasmid lacking transfer functions (oriT+, tra-, mob-) from one E. coli K12 strain to another was investigated in seven sterile microcosms corresponding to different environments. These microcosms were chosen as representative of environments that genetically engineered microorganisms (GEMOs) encounter after accidental release, namely attached biomass in aquatic environments (biofilm), soil, seawater, freshwater, wastewater, mouse gut, and mussel gut, GEMOs survived in the same way as the host strains in all microcosms. Recombinant DNA mobilization occurred in the mouse gut, in sterile soil, and in biofilm. The plasmid transfer rates principally reflected the environmental conditions encountered in each microcosm.

Animals↗
PubMed1996

Structure, energetics, and dynamics of lipid-protein interactions: A molecular dynamics study of the gramicidin A channel in a DMPC bilayer.

The microscopic details of lipid-protein interactions are examined using molecular dynamics simulations of the gramicidin A channel embedded in a fully hydrated dimyristoyl phosphatidylcholine (DMPC) bilayer. A novel construction protocol was used to assemble the initial configurations of the membrane protein complex for the simulations. Three hundred systems were constructed with different initial lipid placement and conformations. Seven systems were simulated with molecular dynamics. One system was simulated for a total of 600 psec, four were simulated for 300 psec, and two for 100 psec. Analysis of the resulting trajectories shows that the bulk solvent-membrane interface region is much broader than traditionally pictured in simplified continuum theories: its width is almost 15 angstroms. In addition, lipid-protein interactions are far more varied, both structurally and energetically, than is usually assumed: the total interaction energy between the gramicidin A and the individual lipids varies from 0 to -50 kcal/mol. The deuterium quadrupolar splittings of the lipid acyl chains calculated from the trajectories are in good agreement with experimental data. The lipid chains in direct contact with the GA are ordered but the effect is not uniform due to the irregular surface of the protein. Energy decompositions shows that the most energetically favorable interactions between lipid and protein involve nearly equal contributions from van der Waals and electrostatic interactions. The tryptophans, located near the bulk-membrane interface, appear to be particularly important in mediating both hydrogen bonding interactions with the lipid glycerol backbone and water and also in forming favorable van der Waals contacts with the hydrocarbon chains. In contrast, the interactions of the leucine residues with the lipids, also located near the interface, are dominated by van der Waals interactions with the hydrocarbon lipid chains.

Biophysics↗
PubMed1996

Denaturation of MM-creatine kinase by sodium dodecyl sulfate.

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm x 11 nm). These results are compatible with recent models of SDS-protein complexes: the "protein decorated micelle structure" or the "necklace structure".

Anilino Naphthalenesulfonates↗
PubMed1996

Differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures.

Mineralization of cartilage and bone requires alkaline phosphatase activity. In order to study the enzymatic properties of bone alkaline phosphatase in bone disease and more particularly in patients with osteoporosis and osteoarthritis, we investigated the solubilization of alkaline phosphatase from primary bone cell cultures derived from human bone explants. To study the release of alkaline phosphatase from membranes, several detergents at a concentration above the critical micellar concentration and cholesterol were used. Solubilized alkaline phosphatase was characterized by enzymatic activity and electrophoresis analysis. Almost all the alkaline phosphatase was solubilized using non-ionic detergent as n-octylglucoside and hecameg. In comparison with initial membranous activity, the solubilized activity was increased by a factor, i.e. 2 +/- 0.05 (SEM, n = 3) (with n-octylglucoside), i.e. 2.1 +/- 0.05 (SEM, n = 3) (with Hecameg). With an ionic detergent (sodium dodecylsulfate), zwitterionic detergent ((cholamido propyl) dimethylammonio 1 propane sulfonate) and cholesterol, a fraction of alkaline phosphatase was resistant to solubilization. Electrophoresis studies showed that released alkaline phosphatase was a glycosylphosphatidylinositol protein (amphipatic form) with 140 kDa as apparent molecular weight. A hydrophilic form was obtained by treatment with a specific lipase. This study showed differential solubilization of osteoblastic alkaline phosphatase from human primary bone cell cultures. Better extractibility and higher activation of this membrane anchored enzyme were obtained with non-ionic detergents.

Alkaline Phosphatase↗
PubMed1996

Structure and dynamics of a proton wire: a theoretical study of H+ translocation along the single-file water chain in the gramicidin A channel.

The rapid translocation of H+ along a chain of hydrogen-bonded water molecules, or proton wire, is thought to be an important mechanism for proton permeation through transmembrane channels. Computer simulations are used to study the properties of the proton wire formed by the single-file waters in the gramicidin A channel. The model includes the polypeptidic dimer, with 22 water molecules and one excess proton. The dissociation of the water molecules is taken into account by the "polarization model" of Stillinger and co-workers. The importance of quantum effects due to the light mass of the hydrogen nuclei is examined with the use of discretized Feynman path integral molecular dynamics simulations. Results show that the presence of an excess proton in the pore orients the single-file water molecules and affects the geometry of water-water hydrogen bonding interactions. Rather than a well-defined hydronium ion OH3+ in the single-file region, the protonated species is characterized by a strong hydrogen bond resembling that of O2H5+. The quantum dispersion of protons has a small but significant effect on the equilibrium structure of the hydrogen-bonded water chain. During classical trajectories, proton transfer between consecutive water molecules is a very fast spontaneous process that takes place in the subpicosecond time scale. The translocation along extended regions of the chain takes place neither via a totally concerted mechanism in which the donor-acceptor pattern would flip over the entire chain in a single step, nor via a succession of incoherent hops between well-defined intermediates. Rather, proton transfer in the wire is a semicollective process that results from the subtle interplay of rapid hydrogen-bond length fluctuations along the water chain. These rapid structural fluctuations of the protonated single file of waters around an average position and the slow movements of the average position of the excess proton along the channel axis occur on two very different time scales. Ultimately, it is the slow reorganization of hydrogen bonds between single-file water molecules and channel backbone carbonyl groups that, by affecting the connectivity and the dynamics of the single-file water chain, also limits the translocation of the proton across the pore.

Biophysical Phenomena↗
PubMed1996

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed.

Amino Acid Sequence↗
PubMed1996

Valence selectivity of the gramicidin channel: a molecular dynamics free energy perturbation study.

The valence selectivity of the gramicidin channel is examined using computer simulations based on atomic models. The channel interior is modeled using a gramicidin-like periodic poly (L,D)-alanine beta-helix. Free energy perturbation calculations are performed to obtain the relative affinity of K+ and Cl- for the channel. It is observed that the interior of the gramicidin channel provides an energetically favorable interaction site for a cation but not for an anion. Relative to solvation in bulk water, the carbonyl CO oxygens can provide a favorable interaction to stabilize K+, whereas the amide NH hydrogens are much less effective in stabilizing Cl-. The results of the calculations demonstrate that, as a consequence of the structural asymmetry of the backbone charge distribution, a K+ cation can partition spontaneously from bulk water to the interior of the gramicidin channel, whereas a Cl- anion cannot.

Calorimetry↗
PubMed1995

Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study.

The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.

Alkaline Phosphatase↗
PubMed1995

Functional interactions in bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding with water.

The light-driven proton pump, bacteriorhodopsin (bR) contains a retinal molecule with a Schiff base moiety that can participate in hydrogen-bonding interactions in an internal, water-containing channel. Here we combine quantum chemistry and molecular mechanics techniques to determine the geometries and energetics of retinal Schiff base-water interactions. Ab initio molecular orbital calculations are used to determine potential surfaces for water-Schiff base hydrogen-bonding and to characterize the energetics of rotation of the C-C single bond distal and adjacent to the Schiff base NH group. The ab initio results are combined with semiempirical quantum chemistry calculations to produce a data set used for the parameterization of a molecular mechanics energy function for retinal. Using the molecular mechanics force field the hydrated retinal and associated bR protein environment are energy-minimized and the resulting geometries examined. Two distinct sites are found in which water molecules can have hydrogen-bonding interactions with the Schiff base: one near the NH group of the Schiff base in a polar region directed towards the extracellular side, and the other near a retinal CH group in a relatively nonpolar region, directed towards the cytoplasmic side.

Bacteriorhodopsins↗
PubMed1995

Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy.

The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions.

Binding Sites↗
PubMed1994

Molecular dynamics simulation of the gramicidin channel in a phospholipid bilayer.

A molecular dynamics simulation of the gramicidin A channel in an explicit dimyristoyl phosphatidylcholine bilayer was generated to study the details of lipid-protein interactions at the microscopic level. Solid-state NMR properties of the channel averaged over the 500-psec trajectory are in excellent agreement with available experimental data. In contrast with the assumptions of macroscopic models, the membrane/solution interface region is found to be at least 12 A thick. The tryptophan side chains, located within the interface, are found to form hydrogen bonds with the ester carbonyl groups of the lipids and with water, suggesting their important contribution to the stability of membrane proteins. Individual lipid-protein interactions are seen to vary from near 0 to -50 kcal/mol. The most strongly interacting conformations are short-lived and have a nearly equal contribution from both van der Waals and electrostatic energies. This approach for performing molecular dynamics simulations of membrane proteins in explicit phospholipid bilayers should help in studying the structure, dynamics, and energetics of lipid-protein interactions.

Gramicidin↗
PubMed1994

Nuclear proteins interacting with DNA and tubulin. Study of the interaction of the High Mobility Group protein 1 with tubulin.

Fractionation of a 0.2 M NaCl nuclear extract from rat liver cells by both tubulin and DNA affinity chromatography steps allowed us to find three polypeptides interacting in vitro with both DNA and tubulin. A 22 kDa polypeptide was identified as a proteolytic fragment of High Mobility Group proteins 1 or 2 (HMG 1 or 2). Purified rat liver HMG 1 immobilized on nitrocellulose was found to bind radioiodinated dimeric tubulin through its central B domain. The C domain of HMG 1 appeared to play a negative role in this association process. Soluble HMG 1 depleted of its C-terminal domain interacted with tubulin immobilized on an agarose gel and with microtubules formed from purified tubulin. In contrast, undigested HMG 1 did not interact with tubulin in these conditions. The modification of HMG 1 with amine by 1-ethyl-3-(dimethylaminopropyl)carbodiimide which caused the neutralization of the C domain carboxyl groups restored the ability of HMG 1 to interact with microtubules. These results show that: (a) HMG 1, through its central B domain, binds to both assembled and non-assembled tubulin in vitro and (b) the C-terminal domain of HMG 1 exerts a negative regulatory action on the interaction.

Animals↗
PubMed1994

Identification of a new transposon Tn5403 in a Klebsiella pneumoniae strain isolated from a polluted aquatic environment.

A Klebsiella pneumoniae strain having mobilization "helper" potential has been isolated from the river Rhine. Analysis of the transconjugants resulting from the mobilization of non-conjugative pBR-type plasmids and RSF1010 derivatives showed that the transfer-helper capacity of the K. pneumoniae strain is related to the presence of a Tn3-like transposable element, Tn5403. This element has been identified and localized in a plasmid.

Base Sequence↗
PubMed1994

A molecular dynamics study of gating in dioxolane-linked gramicidin A channels.

The gating transition of the RR and SS dioxolane ring-linked gramicidin A channels were studied with molecular dynamics simulations using a detailed atomic model. It was found that the probable reaction path, describing the transition of the ring from the exterior to the interior of the channel where it blocked the permeation pathway, involved several steps including the isomerization of the transpeptide plane dihedral angle of Val1. Reaction coordinates along this pathway were defined, and the transition rates between the stable conformers were calculated. It was found, in good accord with experimental observations, that the calculated blocking rate for the RR-linked channel was 280/s with a mean blocking time of 0.04 ms, whereas such blocking did not occur in the case of the SS-linked channel. An important observation is that the resulting lifetime for the blocked state of the RR-linked channel was in good accord with the experimental observations only when the calculations were performed in the presence of a potassium ion inside the channel.

Dioxolanes↗