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B Roux

Publications and source records attributed to B Roux.

At least 19 recordsLinked to original sources
PubMed2004

Insertion of a glycosylphosphatidylinositol-anchored enzyme into liposomes.

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of (3)H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance-about 2.5 nm-between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein.

Alkaline Phosphatase↗
PubMed2003

Role of GPI-anchored enzyme in liposome detergent-resistance.

In this work, we investigated the role of a glycosylphosphatidylinositol (GPI)-anchored protein, the alkaline phosphatase, on the solubilization of detergent-resistant liposomes. In vivo, GPI-anchored proteins are clustered into sphingolipid- and cholesterol-rich membrane domains and this peculiar composition provides cold-detergent-insolubility. To better understand the mechanisms involved in the clustering of these subdomain components, we built a model, namely sphingolipid- and cholesterol-rich liposomes. We show the cold-Triton X-100 resistance of liposomes before and after insertion of GPI-anchored enzyme. When the amount of incorporated enzyme varied, significant changes in membrane stability occurred. Low protein contents into liposomes increased detergent insolubility, whereas high amounts decreased it. Furthermore, significant differences in the detergent-resistance of each lipid were exhibited between liposomes and proteoliposomes. Thus, the enzyme insertion led to a dramatic decrease of cholesterol solubilization, in line with the existence of cholesterol/GPI interactions. Effect of temperature on detergent resistance was also investigated. Liposome solubilization increased with temperature up to a threshold value of 40/45 degrees C. This was also the temperature at which a phase transition of liposome membrane occurred, as evidenced by Laurdan fluorescence. Although the GPI-anchored enzyme insertion modified membrane stability, no change was observed on phase transition. Our work highlights the importance of GPI-anchored proteins in the structure of sphingolipid- and cholesterol-rich membrane domains, in the detergent-insolubility of these peculiar domains, as well as in interaction of GPI proteins with cholesterol.

Alkaline Phosphatase↗
PubMed2001

Energetics of ion conduction through the K+ channel.

K+ channels are transmembrane proteins that are essential for the transmission of nerve impulses. The ability of these proteins to conduct K+ ions at levels near the limit of diffusion is traditionally described in terms of concerted mechanisms in which ion-channel attraction and ion-ion repulsion have compensating effects, as several ions are moving simultaneously in single file through the narrow pore. The efficiency of such a mechanism, however, relies on a delicate energy balance-the strong ion-channel attraction must be perfectly counterbalanced by the electrostatic ion-ion repulsion. To elucidate the mechanism of ion conduction at the atomic level, we performed molecular dynamics free energy simulations on the basis of the X-ray structure of the KcsA K+ channel. Here we find that ion conduction involves transitions between two main states, with two and three K+ ions occupying the selectivity filter, respectively; this process is reminiscent of the 'knock-on' mechanism proposed by Hodgkin and Keynes in 1955. The largest free energy barrier is on the order of 2-3 kcal mol-1, implying that the process of ion conduction is limited by diffusion. Ion-ion repulsion, although essential for rapid conduction, is shown to act only at very short distances. The calculations show also that the rapidly conducting pore is selective.

Bacterial Proteins↗
PubMed2001

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation.

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.

Amino Acid Substitution↗
PubMed2001

Lipid-mediated interactions between intrinsic membrane proteins: dependence on protein size and lipid composition.

The present study is an application of an approach recently developed by the authors for describing the structure of the hydrocarbon chains of lipid-bilayer membranes (LBMs) around embedded protein inclusions ( Biophys. J. 79:2867-2879). The approach is based on statistical mechanical integral equation theories developed for the study of dense liquids. First, the configurations extracted from molecular dynamics simulations of pure LBMs are used to extract the lateral density-density response function. Different pure LBMs composed of different lipid molecules were considered: dioleoyl phosphatidylcholine (DOPC), palmitoyl-oleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), and dimyristoyl phosphatidylcholine (DMPC). The results for the lateral density-density response function was then used as input in the integral equation theory. Numerical calculations were performed for protein inclusions of three different sizes. For the sake of simplicity, protein inclusions are represented as hard smooth cylinders excluding the lipid hydrocarbon core from a small cylinder of 2.5 A radius, corresponding roughly to one aliphatic chain, a medium cylinder of 5 A radius, corresponding to one alpha-helix, and a larger cylinder of 9 A radius, representing a small protein such as the gramicidin channel. The lipid-mediated interaction between protein inclusions was calculated using a closed-form expression for the configuration-dependent free energy. This interaction was found to be repulsive at intermediate range and attractive at short range for two small cylinders in POPC, DPPC, and DMPC bilayers, whereas it oscillates between attractive and repulsive values in DOPC bilayers. For medium size cylinders, it is again repulsive at intermediate range and attractive at short range, but for every model LBM considered here. In the case of a large cylinder, the lipid-mediated interaction was shown to be repulsive for both short and long ranges for the DOPC, POPC, and DPPC bilayers, whereas it is again repulsive and attractive for DMPC bilayers. The results indicate that the packing of the hydrocarbon chains around protein inclusions in LBMs gives rise to a generic (i.e., nonspecific) lipid-mediated interaction which favors the association of two alpha-helices and depends on the lipid composition of the membrane.

Computer Simulation↗
PubMed2001

Framework model for single proton conduction through gramicidin.

This paper describes a framework model for proton conduction through gramicidin; a model designed to incorporate information from molecular dynamics and use this to predict conductance properties. The state diagram describes both motion of an excess proton within the pore as well as the reorientation of waters within the pore in the absence of an excess proton. The model is constructed as the diffusion limit of a random walk, allowing control over the boundary behavior of trajectories. Simple assumptions about the boundary behavior are made, which allow an analytical solution for the proton current and conductance. This is compared with corresponding expressions from statistical mechanics. The random walk construction allows diffusing trajectories underlying the model to be simulated in a simple way. Details of the numerical algorithm are described.

Algorithms↗
PubMed2001

Extracellular blockade of K(+) channels by TEA: results from molecular dynamics simulations of the KcsA channel.

TEA is a classical blocker of K(+) channels. From mutagenesis studies, it has been shown that external blockade by TEA is strongly dependent upon the presence of aromatic residue at Shaker position 449 which is located near the extracellular entrance to the pore (Heginbotham, L., and R. MacKinnon. 1992. Neuron. 8:483-491). The data suggest that TEA interacts simultaneously with the aromatic residues of the four monomers. The determination of the 3-D structure of the KcsA channel using X-ray crystallography (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) has raised some issues that remain currently unresolved concerning the interpretation of these observations. In particular, the center of the Tyr82 side chains in KcsA (corresponding to position 449 in Shaker) forms a square of 11.8-A side, a distance which is too large to allow simultaneous interactions of a TEA molecule with the four aromatic side chains. In this paper, the external blockade by TEA is explored by molecular dynamics simulations of an atomic model of KcsA in an explicit phospholipid bilayer with aqueous salt solution. It is observed, in qualitative accord with the experimental results, that TEA is stable when bound to the external side of the wild-type KcsA channel (with Tyr82), but is unstable when bound to a mutant channel in which the tyrosine residue has been substituted by a threonine. The free energy profile of TEA relative to the pore is calculated using umbrella sampling simulations to characterize quantitatively the extracellular blockade. It is found, in remarkable agreement with the experiment, that the TEA is more stably bound by 2.3 kcal/mol to the channel with four tyrosine residues. In the case of the wild-type KcsA channel, TEA (which has the shape of a flattened oblate spheroid) acts as an ideal plug blocking the pore. In contrast, it is considerably more off-centered and tilted in the case of the mutant channel. The enhanced stability conferred by the tyrosine residues does not arise from Pi-cation interactions, but appears to be due to differences in the hydration structure of the TEA. Finally, it is shown that the experimentally observed voltage dependence of TEA block, which is traditionally interpreted in terms of the physical position of the TEA along the axis of the pore, must arise indirectly via coupling with the ions in the pore.

Bacterial Proteins↗
PubMed2000

Anchoring of a monotopic membrane protein: the binding of prostaglandin H2 synthase-1 to the surface of a phospholipid bilayer.

Prostaglandin H2 synthases (PGHS-1 and -2) are monotopic peripheral membrane proteins that catalyse the synthesis of prostaglandins in the arachidonate cascade. Picot et al. (1994) proposed that the enzyme is anchored to one leaflet of the bilayer by a membrane anchoring domain consisting of a right-handed spiral of amphipathic helices (residues 73-116) forming a planar motif. Two different computational approaches are used to examine the association of the PGHS-1 membrane anchoring domain with a membrane via the proposed mechanism. The electrostatic contribution to the free energy of solvation is obtained by solving numerically the finite-difference Poisson equation for the protein attached to a membrane represented as a planar slab of low dielectric. The nonpolar cavity formation and van der Waals contributions to the solvation free energy are assumed to be proportional to the water accessible surface area. Based on the optimum position determined from the continuum solvent model, two atomic models of the PGHS-1 anchoring domain associated with an explicit dimyristoylphosphatidylcholine (DMPC) bilayer differing by the thickness of the membrane bilayer were constructed. A total of 2 ns molecular dynamics simulation were performed to study the details of lipid-protein interactions at the microscopic level. In the simulations the lipid hydrocarbon chains interacting with the anchoring domain assume various shapes, suggesting that the plasticity of the membrane is significant. The hydrophobic residues in the membrane side of the helices interact with the hydrophobic membrane core, while the positively charged residues interact with the lipid polar headgroups to stabilize the anchoring of the membrane domain to the upper half of the bilayer. The phosphate headgroup of one DMPC molecule disposed at the center of the spiral formed by helices A, B, C and D interacts strongly with Arg120, a residue on helix D that has previously been identified as being important in the activity of PGHS-1. In the full enzyme structure, this position corresponds to the entrance of a long hydrophobic channel leading to the cyclooxygenase active site. These observations provide insights into the association of the arachidonic acid substrate to the cyclooxygenase active site of PGHS-1.

Binding Sites↗
PubMed2000

Activity increase after extraction of alkaline phosphatase from human osteoblastic membranes by nonionic detergents: influence of age and sex.

The solubilization of alkaline phosphatase (AP) from osteoblastic cell membranes obtained from human primary bone cell cultures was studied according to the age and sex of the donors (17 females, 11 males; age range: 2-77 years). Cell membranes were treated by non-ionic (n-octyl beta-D-glucopyranoside, OG), ionic or zwitterionic detergents, then centrifuged. When OG was used almost all the AP was solubilized. AP activity in supernatant of solubilization was compared to the activity of the suspension before centrifugation. The activity ratio (AR) increased in function of age for subjects between 65 and 74. Neither total nor specific AP activities were influenced by age or sex. Electrophoresis studies showed that the AP released was a GPI (glycosyl phosphatidylinositol)-anchored protein, amphipathic form, with 140 kDa as apparent molecular mass. The activity change of AP in the presence of OG may result from age-related modifications either in the AP structure or in the constituents of the plasma membranes (proteins or phospholipids).

Adolescent↗
PubMed2000

A Grand Canonical Monte Carlo-Brownian dynamics algorithm for simulating ion channels.

A computational algorithm based on Grand Canonical Monte Carlo (GCMC) and Brownian Dynamics (BD) is described to simulate the movement of ions in membrane channels. The proposed algorithm, GCMC/BD, allows the simulation of ion channels with a realistic implementation of boundary conditions of concentration and transmembrane potential. The method is consistent with a statistical mechanical formulation of the equilibrium properties of ion channels (; Biophys. J. 77:139-153). The GCMC/BD algorithm is illustrated with simulations of simple test systems and of the OmpF porin of Escherichia coli. The approach provides a framework for simulating ion permeation in the context of detailed microscopic models.

Algorithms↗
PubMed2000

A combined molecular dynamics and diffusion model of single proton conduction through gramicidin.

We develop a model for proton conduction through gramicidin based on the molecular dynamics simulations of Pomès and Roux (Biophys. J. 72:A246, 1997). The transport of a single proton through the gramicidin pore is described by a potential of mean force and diffusion coefficient obtained from the molecular dynamics. In addition, the model incorporates the dynamics of a defect in the hydrogen bonding structure of pore waters without an excess proton. Proton entrance and exit were not simulated by the molecular dynamics. The single proton conduction model includes a simple representation of these processes that involves three free parameters. A reasonable value can be chosen for one of these, and the other two can be optimized to yield a good fit to the proton conductance data of, Ann. N.Y. Acad. Sci. 339:8-20) for pH > or = 1.7. A sensitivity analysis shows the significance of this fit.

Biophysics↗
PubMed2000

Lipid-mediated interactions between intrinsic membrane proteins: a theoretical study based on integral equations.

This study of lipid-mediated interactions between proteins is based on a theory recently developed by the authors for describing the structure of the hydrocarbon chains in the neighborhood of a protein inclusion embedded in a lipid membrane [Lagüe et al., Farad. Discuss. 111:165-172, 1998]. The theory involves the hypernetted chain integral equation formalism for liquids. The exact lateral density-density response function of the hydrocarbon core, extracted from molecular dynamics simulations of a pure dipalmitoylphosphatidylcholine bilayer based on an atomic model, is used as input. For the sake of simplicity, protein inclusions are modeled as hard repulsive cylinders. Numerical calculations were performed with three cylinder sizes: a small cylinder of 2.5-A radius, corresponding roughly to an aliphatic chain; a medium cylinder of 5-A radius, corresponding to a alpha-helical polyalanine protein; and a large cylinder of 9-A radius, representing a small protein, such as the gramicidin channel. The calculations show that the average hydrocarbon density is perturbed over a distance of 20-25 A from the edge of the cylinder for every cylinder size. The lipid-mediated protein-protein effective interaction is calculated and is shown to be nonmonotonic. In the case of the small and the medium cylinders, the lipid-mediated effective interaction of two identical cylinders is repulsive at an intermediate range but attractive at short range. At contact, there is a free energy of -2k(B)T for the 2.5-A-radius cylinder and -9k(B)T for the 5-A-radius cylinder, indicating that the association of two alpha-helices of both sizes is favored by the lipid matrix. In contrast, the effective interaction is repulsive at all distances in the case of the large cylinder. Results were obtained with two integral equations theories: hypernetted chain and Percus-Yevick. For the two theories, all results are qualitatively identical.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2000

Molecular dynamics of the KcsA K(+) channel in a bilayer membrane.

Molecular dynamics (MD) simulations of an atomic model of the KcsA K(+) channel embedded in an explicit dipalmitoylphosphatidylcholine (DPPC) phospholipid bilayer solvated by a 150 mM KCl aqueous salt solution are performed and analyzed. The model includes the KcsA K(+) channel, based on the recent crystallographic structure of, Science. 280:69-77), 112 DPPC, K(+) and Cl(-) ions, as well as over 6500 water molecules for a total of more than 40,000 atoms. Three K(+) ions are explicitly included in the pore. Two are positioned in the selectivity filter on the extracellular side and one in the large water-filled cavity. Different starting configurations of the ions and water molecules in the selectivity filter are considered, and MD trajectories are generated for more than 4 ns. The conformation of KcsA is very stable in all of the trajectories, with a global backbone root mean square (RMS) deviation of less than 1.9 A with respect to the crystallographic structure. The RMS atomic fluctuations of the residues surrounding the selectivity filter on the extracellular side of the channel are significantly lower than those on the intracellular side. The motion of the residues with aromatic side chains surrounding the selectivity filter (Trp(67), Trp(68), Tyr(78), and Tyr(82)) is anisotropic with the smallest RMS fluctuations in the direction parallel to the membrane plane. A concerted dynamic transition of the three K(+) ions in the pore is observed, during which the K(+) ion located initially in the cavity moves into the narrow part of the selectivity filter, while the other two K(+) ions move toward the extracellular side. A single water molecule is stabilized between each pair of ions during the transition, suggesting that each K(+) cation translocating through the narrow pore is accompanied by exactly one water molecule, in accord with streaming potential measurements (, Biophys. J. 55:367-371). The displacement of the ions is coupled with the structural fluctuations of Val(76) and Gly(77), in the selectivity filter, as well as the side chains of Glu(71), Asp(80), and Arg(89), near the extracellular side. Thus the mechanical response of the channel structure at distances as large as 10-20 A from the ions in the selectivity filter appears to play an important role in the concerted transition.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1999

Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa.

Alkaline phosphatase (EC 3.1.3.1) from bovine intestinal mucosa (BIAP) is an homodimeric metalloenzyme, containing one Mg2+ and two Zn2+ ions in each active site. ApoBIAP, prepared using ion-chelating agents, exhibited a dramatic decrease of its hydrolase activity, concomittant to conformational changes in its quaternary structure. By rate-zonal centrifugation and electrophoresis, we demonstrated, for the first time, that the loss of divalent ions leads to some monomerization process for a metal-depleted alkaline phosphatase. Divalent ions are also involved in the secondary and tertiary structures. Metal-depletion induced more exposure of some Trp residues and hydrophobic regions to the solvent (as proved by intrinsic and ANS fluorescences). These changes might correspond to the disappearance of alpha-helices and/or turns with a concomittant appearance of unordered structures and beta-sheets (as probed by FTIR spectroscopy). For BIAP, three steps of temperature-induced changes were exhibited, while for apoBIAP, only one step was exhibited at 55 degrees C. Our work on BIAP showed two main differences with alkaline phosphatase from Escherichia coli. The loss of the divalent ions induces protein monomerization and the total recovery of enzyme activity by divalent ion addition to apoBIAP was not obtained.

Alkaline Phosphatase↗
PubMed1999

A FTIR spectroscopy evidence of the interactions between wheat germ agglutinin and N-acetylglucosamine residues.

Wheat germ agglutinin (WGA), a lectin binding a N-acetyl-D-neuraminic acid (NeuNAc) and/or N-acetyl-D-glucosamine (GlcNAc) group, was studied by Fourier transform infrared (FTIR) spectroscopy. Deconvolution of the FTIR spectrum of WGA alone indicated the presence of few alpha-helices and beta-sheets, in contrast to many other lectins. These results agree with previous WGA crystal data. The WGA conformational changes, induced by GlcNAc-bearing liposomes or GlcNAc oligomers, were studied by infrared differential spectroscopy. The GlcNAc binding to WGA resulted in a decrease of turns and alpha-helices and a concomitant appearance of beta-sheets, inducing more or less peptidic N-H deuteration.

Acetylglucosamine↗
PubMed1999

The cavity and pore helices in the KcsA K+ channel: electrostatic stabilization of monovalent cations.

The electrostatic influence of the central cavity and pore alpha helices in the potassium ion channel from Streptomyces lividans (KcsA K+ channel) was analyzed by solving the finite difference Poisson equation. The cavity and helices overcome the destabilizing influence of the membrane and stabilize a cation at the membrane center. The electrostatic effect of the pore helices is large compared to that described for water-soluble proteins because of the low dielectric membrane environment. The combined contributions of the ion self-energy and the helix electrostatic field give rise to selectivity for monovalent cations in the water-filled cavity. Thus, the K+ channel uses simple electrostatic principles to solve the fundamental problem of ion destabilization by the cell membrane lipid bilayer.

Bacterial Proteins↗
PubMed1999

Implicit solvent models.

Implicit solvent models for biomolecular simulations are reviewed and their underlying statistical mechanical basis is discussed. The fundamental quantity that implicit models seek to approximate is the solute potential of mean force, which determines the statistical weight of solute conformations, and which is obtained by averaging over the solvent degrees of freedom. It is possible to express the total free energy as the reversible work performed in two successive steps. First, the solute is inserted in the solvent with zero atomic partial charges; second, the atomic partial charges of the solute are switched from zero to their full values. Consequently, the total solvation free energy corresponds to a sum of non-polar and electrostatic contributions. These two contributions are often approximated by simple geometrical models (such as solvent exposed area models) and by macroscopic continuum electrostatics, respectively. One powerful route is to approximate the average solvent density distribution around the solute, i.e. the solute-solvent density correlation functions, as in statistical mechanical integral equations. Recent progress with semi-analytical approximations makes continuum electrostatics treatments very efficient. Still more efficient are fully empirical, knowledge-based models, whose relation to explicit solvent treatments is not fully resolved, however. Continuum models that treat both solute and solvent as dielectric continua are also discussed, and the relation between the solute fluctuations and its macroscopic dielectric constant(s) clarified.

Journal Article↗