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B Roux

Publications and source records attributed to B Roux.

At least 37 records · Page 2Linked to original sources
PubMed1999

Optimized atomic radii for protein continuum electrostatics solvation forces.

Recently, we presented a Green's function approach for the calculation of analytic continuum electrostatic solvation forces based on numerical solutions of the finite-difference Poisson-Botzmann (FDPB) equation [Im et al., Comp. Phys. Comm. 111 (1998) 59]. In this treatment the analytic forces were explicitly defined as the first derivative of the FDPB continuum electrostatic free energy with respect to the coordinates of the solute atoms. A smooth intermediate region for the solute-solvent dielectric boundary needed to be introduced to avoid abrupt discontinuous variations in the solvation free energy and forces as a function of the atomic positions. In the present paper we extend the set of optimized radii, which was previously parametrized from molecular dynamics free energy simulations of the 20 standard amino acids with explicit solvent molecules [Nina et al., J. Phys. Chem. 101 (1997) 5239], to yield accurate solvation free energy by taking the influence of the smoothed dielectric region into account.

Journal Article↗
PubMed1999

The binding site of sodium in the gramicidin A channel.

The available information concerning the structure and location of the main binding site for sodium in the gramicidin A channel is reviewed and discussed. Results from molecular dynamics simulations using an atomic model of the channel embedded in a lipid bilayer are compared with experimental observations. The combined information from experiment and simulation suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the centre of the dimer channel, although the motion along the axis could be as large as 1 to 2 A. In the binding site, the sodium ion is lying off axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deflection from the ion-free channel structure.

Anti-Bacterial Agents↗
PubMed1999

Statistical mechanical equilibrium theory of selective ion channels.

A rigorous statistical mechanical formulation of the equilibrium properties of selective ion channels is developed, incorporating the influence of the membrane potential, multiple occupancy, and saturation effects. The theory provides a framework for discussing familiar quantities and concepts in the context of detailed microscopic models. Statistical mechanical expressions for the free energy profile along the channel axis, the cross-sectional area of the pore, and probability of occupancy are given and discussed. In particular, the influence of the membrane voltage, the significance of the electric distance, and traditional assumptions concerning the linearity of the membrane electric field along the channel axis are examined. Important findings are: 1) the equilibrium probabilities of occupancy of multiply occupied channels have the familiar algebraic form of saturation properties which is obtained from kinetic models with discrete states of denumerable ion occupancy (although this does not prove the existence of specific binding sites; 2) the total free energy profile of an ion along the channel axis can be separated into an intrinsic ion-pore free energy potential of mean force, independent of the transmembrane potential, and other contributions that arise from the interfacial polarization; 3) the transmembrane potential calculated numerically for a detailed atomic configuration of the gramicidin A channel embedded in a bilayer membrane with explicit lipid molecules is shown to be closely linear over a distance of 25 A along the channel axis. Therefore, the present analysis provides some support for the constant membrane potential field approximation, a concept that has played a central role in the interpretation of flux data based on traditional models of ion permeation. It is hoped that this formulation will provide a sound physical basis for developing nonequilibrium theories of ion transport in selective biological channels.

Biological Transport↗
PubMed1999

Simulation analysis of the retinal conformational equilibrium in dark-adapted bacteriorhodopsin.

In dark-adapted bacteriorhodopsin (bR) the retinal moiety populates two conformers: all-trans and (13,15)cis. Here we examine factors influencing the thermodynamic equilibrium and conformational transition between the two forms, using molecular mechanics and dynamics calculations. Adiabatic potential energy mapping indicates that whereas the twofold intrinsic torsional potentials of the C13==C14 and C15==N16 double bonds favor a sequential torsional pathway, the protein environment favors a concerted, bicycle-pedal mechanism. Which of these two pathways will actually occur in bR depends on the as yet unknown relative weight of the intrinsic and environmental effects. The free energy difference between the conformers was computed for wild-type and modified bR, using molecular dynamics simulation. In the wild-type protein the free energy of the (13,15)cis retinal form is calculated to be 1.1 kcal/mol lower than the all-trans retinal form, a value within approximately kBT of experiment. In contrast, in isolated retinal the free energy of the all-trans state is calculated to be 2.1 kcal/mol lower than (13,15)cis. The free energy differences are similar to the adiabatic potential energy differences in the various systems examined, consistent with an essentially enthalpic origin. The stabilization of the (13,15)cis form in bR relative to the isolated retinal molecule is found to originate from improved protein-protein interactions. Removing internal water molecules near the Schiff base strongly stabilizes the (13,15)cis form, whereas a double mutation that removes negative charges in the retinal pocket (Asp85 to Ala; Asp212 to Ala) has the opposite effect.

Bacteriorhodopsins↗
PubMed1999

Ectoplasmic insertion of a glycosylphosphatidylinositol-anchored protein in glycosphingolipid- and cholesterol-containing phosphatidylcholine vesicles.

Glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins are proposed to be clustered in membrane microdomains enriched in cholesterol and glycosphingolipids (GlySphs). We have prepared biomimetic membranes in order to study the possible phenomena of surface aggregation of these membrane components. Phosphatidylcholine liposomes were treated by octylglucoside to insert a glycosyl-PtdIns-protein, alkaline phosphatase (ALP), some cholesterol, and a GlySph, the lactocerebroside. The association of these compounds was shown by centrifugation on a density gradient. The presence of ALP on the surface of the vesicles was shown by the action of a phospholipase, and the presence of the lactocerebroside was shown by the use of a galactose-specific tetravalent lectin. Our data show that total alkaline phosphatase and half to total lactocerebroside were ectoplasmically inserted in the vesicles membrane. In addition, we observed that the presence of small amounts of ALP in the liposomes led to significant changes in membrane stability with regard to detergent, as shown by the changes in the solubilization process monitored by turbidimetry. Furthermore, we have built an original method to study the cohesion of the vesicles membrane, in which some magnesium ions were trapped in the luminal space of the liposomes during several days. The ALP is magnesium-dependent for its catalytic activity and was inhibited after incubation of ALP-containing liposomes in a magnesium-free buffer. The ALP activity was restored by the addition of detergent to the liposomes, due to the release of the luminal magnesium ions. Surface aggregation phenomena will now be investigated by atomic force microscopy.

Alkaline Phosphatase↗
PubMed1998

Molecular dynamics study of calbindin D9k in the apo and singly and doubly calcium-loaded states.

Molecular dynamics simulations based on detailed atomic models are used to examine the structure and dynamics of calbindin D9k, a protein possessing a pair of EF-hands able to bind two calcium ions in a cooperative fashion. Trajectories for the apo and singly (in the C-terminal binding site) and doubly loaded structures are generated and analyzed. Each system is solvated in a 27 A radius sphere of 2,285 explicit water molecules. The influence of the remaining bulk is incorporated through a stochastic boundary potential including a solvent reaction field. Long-range electrostatic interactions are treated with a special method and are not truncated. The average structural and dynamic properties upon calcium binding are studied at the atomic level to gain insight into the cooperative interactions between the two binding sites. Results from the trajectories are compared with data from nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. NMR 15N and 13C(alpha) backbone relaxation order parameters and crystallographic B-factors are calculated. Generally, there is a good qualitative agreement between calculated and observed properties. Results confirm that the doubly loaded state is closer, both structurally and dynamically, to the singly loaded state than either of these is to the apo state. It is observed that both hydrogen bonding and the packing of nonpolar side chains contribute to the coupling between the calcium binding sites. Two backbone-to-backbone hydrogen bonds linking the calcium-binding EF-hands (Leu23-0 ... HN-Val61 and Val61-O ... HN-Leu23) are sensitive to the state of occupancy. Residues Leu23 and Val61 exhibit the smallest rms fluctuations of the entire protein in the D state. In addition, the van der Waals interaction of Val61 with the rest of the protein varies with the calcium-binding state.

Binding Sites↗
PubMed1998

Free energy profiles for H+ conduction along hydrogen-bonded chains of water molecules.

The molecular mechanism for proton conduction along hydrogen-bonded chains, or "proton wires," is studied with free energy simulations. The complete transport of a charge along a proton wire requires two complementary processes: 1) translocation of an excess proton (propagation of an ionic defect), and 2) reorientation of the hydrogen-bonded chain (propagation of a bonding defect). The potential of mean force profile for these two steps is computed in model systems comprising a single-file chain of nine dissociable and polarizable water molecules represented by the PM6 model of Stillinger and co-workers. Results of molecular dynamics simulations with umbrella sampling indicate that the unprotonated chain is preferably polarized, and that the inversion of its total dipole moment involves an activation free energy of 8 kcal/mol. In contrast, the rapid translocation of an excess H+ across a chain extending between two spherical solvent droplets is an activationless process. These results suggest that the propagation of a bonding defect constitutes a limiting step for the passage of several protons along single-file chains of water molecules, whereas the ionic translocation may be fast enough to occur within the lifetime of transient hydrogen-bonded water chains in biological membranes.

Biophysical Phenomena↗
PubMed1998

Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic resonance (NMR) and polarized attenuated total internal reflectance Fourier transform infrared (PATIR-FTIR) properties of melittin are calculated from the trajectory to characterize the orientation of the peptide relative to the bilayer. The residue Lys7 located in the hydrophobic moiety of the helix and residues Lys23, Arg24, Gln25, and Gln26 at the C-terminus hydrophilic form hydrogen bonds with water molecules and with the ester carbonyl groups of the lipids, suggesting their important contribution to the stability of the helix in the bilayer. Lipid acyl chains are closely packed around melittin, contributing to the stable association with the membrane. Calculated density profiles and order parameters of the lipid acyl chains averaged over the molecular dynamics trajectory indicate that melittin has effects on both layers of the membrane. The presence of melittin in the upper layer causes a local thinning of the bilayer that favors the penetration of water through the lower layer. The energetic factors involved in the association of melittin at the membrane surface are characterized using an implicit mean-field model in which the membrane and the surrounding solvent are represented as structureless continuum dielectric material. The results obtained by solving the Poisson-Bolztmann equation numerically are in qualitative agreement with the detailed dynamics. The influence of the protonation state of the N-terminus of melittin is examined. After 600 ps, the N-terminus of melittin is protonated and the trajectory is continued for 400 ps, which leads to an important penetration of water molecules into the bilayer. These observations provide insights into how melittin interacts with membranes and the mechanism by which it enhances their lysis.

Amino Acid Sequence↗
PubMed1998

Protein inclusion in lipid membranes: a theory based on the hypernetted chain integral equation.

A theory for describing the structure of the hydrocarbon chains around a protein inclusion embedded in a lipid bilayer is developed on the basis of the hypernetted chain integral equation formalism for liquids. The exact lateral density-density response function of the hydrocarbon core, which is extracted from a molecular dynamics simulation of a pure lipid bilayer, is used as input to the theory. Numerical calculations show that the average lipid order is perturbed over a distance of 25 to 30 A around a hard repulsive cylinder of 5 A radius representing an alpha-helical polyleucine protein inclusion. The lipid-mediated protein-protein interaction is calculated and is shown to be non-monotonic, being repulsive at an intermediate range but attractive at short range. It is found that the lipid matrix contributes a free energy well of 8 kBT to the association of two cylindrical inclusions.

Cell Membrane↗
PubMed1997

High-resolution polypeptide structure in a lamellar phase lipid environment from solid state NMR derived orientational constraints.

BACKGROUND: Solid-state nuclear magnetic resonance (NMR) spectroscopy provides novel structural constraints from uniformly aligned samples. These orientational constraints orient specific atomic sites with respect to the magnetic field direction and the unique molecular axis of alignment. Solid-state NMR is uniquely and ideally suited for providing such structural constraints on polypeptides and proteins in a lamellar phase lipid environment. Membrane protein structure represents a great challenge for structural biologists; a new approach for characterizing high resolution three-dimensional structure in such an environment is needed. RESULTS: The optimal use of orientational constraints for defining three-dimensional structures is demonstrated with the elucidation of the gramicidin A channel structure at high resolution. Initial structures are refined against both the experimental constraints and the CHARMM energy using a novel simulated-annealing protocol to define torsion angle solutions with an error bar of approximately +/- 5 degrees. CONCLUSIONS: This analysis results in the determination of a high-resolution, time averaged structure of gramicidin A obtained in a lipid bilayer environment above the gel-to-liquid crystalline phase transition temperature. It is demonstrated that solid-state NMR can be used to establish polypeptide, and potentially protein, structures in such an environment. Furthermore, this high-resolution structure is demonstrated to provide new insights into polypeptide function. For the gramicidin A channel the roles of the indole groups that facilitate ion transport and details of the cation solvation environment provided by the amide oxygens are characterized.

Amino Acid Sequence↗
PubMed1997

Detergent-mediated reconstitution of a glycosyl-phosphatidylinositol-protein into liposomes.

A three-step detergent-mediated reconstitution has been applied to the incorporation of a glycosyl-phosphatidylinositol-protein into liposomes. The protein studied was alkaline phosphatase from bovine intestine. Liposomes prepared by dialysis were treated with various amounts of two detergents, either n-octyl beta-D-glucoside or Triton X-100. At different steps of the solubilization process, protein was added and the detergent was removed by hydrophobic resins. The most efficient reconstitutions were obtained with an octyl glucoside concentration corresponding to the onset of liposome solubilization and with a Triton X-100 concentration leading to partial solubilization of the liposomes. The involvement of the glycosyl-phosphatidylinositol anchor in alkaline phosphatase reconstitution was demonstrated by the inability of phosphoinositol-specific phospholipase-C-hydrolysed alkaline phosphatase to incorporate into liposomes. Between 70-85% of the protein associated with liposomes were anchored in the outer leaflet of the bilayer, oriented towards the outside of the liposome. The remainder was trapped within the lumen of the liposomes.

Alkaline Phosphatase↗
PubMed1997

Kinetics of peptide folding: computer simulations of SYPFDV and peptide variants in water.

The folding of Ser-Tyr-Pro-Phe-Asp-Val (SYPFDV), and sequence variants of this peptide (SYPYD and SYPFD) are studied computationally in an explicit water environment. An atomically detailed model of the peptide is embedded in a sphere of TIP3P water molecules and its optimal structure is computed by simulated annealing. At distances from the peptide that are beyond a few solvation shells, a continuum solvent model is employed. The simulations are performed using a mean field approach that enhances the efficiency of sampling peptide conformations. The computations predict a small number of conformations as plausible folded structures. All have a type VI turn conformation for the peptide backbone, similar to that found using NMR. However, some of the structures differ from the experimentally proposed ones in the packing of the proline ring with the aromatic residues. The second most populated structure has, in addition to a correctly folded backbone, the same hydrophobic packing as the conformation measured by NMR. Our simulations suggest a kinetic mechanism that consists of three separate stages. The time-scales associated with these stages are distinct and depend differently on temperature. Electrostatic interactions play an initial role in guiding the peptide chain to a roughly correct structure as measured by the end-to-end distance. At the same time or later the backbone torsions rearrange due to local tendency of the proline ring to form a turn: this step depends on solvation forces and is helped by loose hydrophobic interactions. In the final step, hydrophobic residues pack against each other. We also show the existence of an off the pathway intermediate, suggesting that even in the folding of a small peptide "misfolded" structures can form. The simulations clearly show that parallel folding paths are involved. Our findings suggest that the process of peptide folding shares many of the features expected for the significantly larger protein molecules.

Computer Simulation↗
PubMed1997

Human tissue non-specific alkaline phosphatases: sugar-moiety-induced enzymic and antigenic modulations and genetic aspects.

To investigate the possible role(s) of glycans in human tissue non-specific alkaline phosphatase (TNAP) activity, the iso-enzymes were purified and treated with various exo- and endo-glycosidases. Catalytic activity, oligomerization, conformation and immunoreactivity of the modified TNAPs were evaluated. All TNAPs proved to be N-glycosylated, and only the liver isoform (LAP) is not O-glycosylated. Usually, the kidney (KAP) and bone (BAP) isoenzymes are similar and cannot be clearly discriminated. Differences between the immunoreactivity of KAP/BAP and LAP with a BAP antibody were mainly attributed to the N-glycosylated moieties of the TNAPs. In addition, elimination of O-glycosylations moderately affects the TNAP reactivity. Interestingly, N-glycosylation is absolutely essential for TNAP activity, but not for that of the placental or intestinal enzymes. According to the deduced amino acid sequence of TNAP cDNA, Asn-213 is a possible N-glycosylation site, and our present findings suggest that this sugar chain plays a key role in enzyme regulation. With regard to the oligomeric state of alkaline phosphatase (AP) isoforms, the dimer/tetramer equilibrium is dependent on the deglycosylation of glycosyl-phosphatidylinositol(GPI)-free APs, but not GPI-linked APs. This equilibrium does not affect the AP conformation as observed with CD. With regard to TNAPs, no data were available on the gene expression or nature of the 5'-non-translated leader exon of human KAP, as opposed to BAP and LAP genes. cDNA sequencing revealed that cortex/medulla KAP is genetically related to BAP, and medulla KAP to LAP.

Alkaline Phosphatase↗
PubMed1997

Influence of the membrane potential on the free energy of an intrinsic protein.

A modified Poisson-Boltzmann equation is developed from statistical mechanical considerations to describe the influence of the transmembrane potential on macromolecular systems. Using a Green's function formalism, the electrostatic free energy of a protein associated with the membrane is expressed as the sum of three terms: a contribution from the energy required to charge the system's capacitance, a contribution corresponding to the interaction of the protein charges with the membrane potential, and a contribution corresponding to a voltage-independent reaction field free energy. The membrane potential, which is due to the polarization interface, is calculated in the absence of the protein charges, whereas the reaction field is calculated in the absence of transmembrane potential. Variations in the capacitive energy associated with typical molecular processes are negligible under physiological conditions. The formulation of the theory is closely related to standard algorithms used to solve the Poisson-Boltzmann equation and only small modifications to current source codes are required for its implementation. The theory is illustrated by examining the voltage-dependent membrane insertion of a simple polyalanine alpha-helix and by computing the electrostatic potential across a 60-A-diameter sphere meant to represent a large intrinsic protein.

Algorithms↗