Computer simulation of ion channel gating: the M(2) channel of influenza A virus in a lipid bilayer.
(33/927)
The transmembrane fragment of the influenza virus M(2) protein forms a homotetrameric channel that transports protons. In this paper, we use molecular dynamics simulations to help elucidate the mechanism of channel gating by four histidines that occlude the channel lumen in the closed state. We test two competing hypotheses. In the "shuttle" mechanism, the delta nitrogen atom on the extracellular side of one histidine is protonated by the incoming proton, and, subsequently, the proton on the epsilon nitrogen atom is released on the opposite side. In the "water-wire" mechanism, the gate opens because of electrostatic repulsion between four simultaneously biprotonated histidines. This allows for proton transport along the water wire that penetrates the gate. For each system, composed of the channel embedded in a hydrated phospholipid bilayer, a 1.3-ns trajectory was obtained. It is found that the states involved in the shuttle mechanism, which contain either single-protonated histidines or a mixture of single-protonated histidines plus one biprotonated residue, are stable during the simulations. Furthermore, the orientations and dynamics of water molecules near the gate are conducive to proton transfer. In contrast, the fully biprotonated state is not stable. Additional simulations show that if only two histidines are biprotonated, the channel deforms but the gate remains closed. These results support the shuttle mechanism but not the gate-opening mechanism of proton gating in M(2). (+info)
A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes.
(34/927)
High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC >> DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component. (+info)
Kinetics of amphiphile association with two-phase lipid bilayer vesicles.
(35/927)
We examined the consequences of membrane heterogeneity for the association of a simple amphiphilic molecule with phospholipid vesicles with solid-liquid and liquid-liquid phase coexistence. To address this problem we studied the association of a single-chain, fluorescent amphiphile with dimyristoylphosphatidylcholine (DMPC) vesicles containing varying amounts of cholesterol. DMPC bilayers containing 15 mol% cholesterol show a region of solid-liquid-ordered (s-l(o)) coexistence below the T(m) of pure DMPC (23.9 degrees C) and a region of liquid-disordered-liquid-ordered coexistence (l(d)-l(o)) above the T(m). We first examined equilibrium binding and kinetics of amphiphile insertion into single-phase vesicles (s, l(d), and l(o) phase). The data obtained were then used to predict the behavior of the equivalent process in a two-phase system, taking into account the fractions of phases present. Next, the predicted kinetics were compared to experimental kinetics obtained from a two-phase system. We found that association of the amphiphile with lipid vesicles is not influenced by the existence of l(d)-l(o) phase boundaries but occurs much more slowly in the s-l(o) phase coexistence region than expected on the basis of phase composition. (+info)
Acidic phospholipid bicelles: a versatile model membrane system.
(36/927)
With the aim of establishing acidic bicellar solutions as a useful membrane model system, we have used deuterium NMR spectroscopy to investigate the properties of dimyristoyl/dihexanoylphosphatidylcholine (DMPC/DHPC) bicelles containing 25% (w/w in H(2)O) of either dimyristoylphosphatidylserine (DMPS) or dimyristoylphosphatidylglycerol (DMPG). The addition of the acidic lipid component to this lyotropic liquid crystalline system reduces its range of stability because of poor miscibility of the two dimyristoylated phospholipids. Compared to the neutral bicelles, which are stable at pH 4 to pH 7, acidic bicelles are stable only from pH 5.5 to pH 7. Solid-state deuterium NMR analysis of d(54)-DMPC showed similar ordering in neutral and acidic bicelles. Fully deuterated DMPS or DMPG is ordered in a way similar to that of DMPC. Study of the binding of the myristoylated N-terminal 14-residue peptide mu-GSSKSKPKDPSQRR from pp60(nu-src) to both neutral and acidic bicelles shows the utility of these novel membrane mimetics. (+info)
Ideally amphipathic beta-sheeted peptides at interfaces: structure, orientation, affinities for lipids and hemolytic activity of (KL)(m)K peptides.
(37/927)
Designed to model ideally amphipathic beta-sheets, the minimalist linear (KL)(m)K peptides (m=4-7) were synthesized and proved to form stable films at the air/water interface, they insert into compressed dimyristoylphosphatidylcholine monolayers and interact with egg phosphatidylcholine vesicles. Whatever the interface or the lateral pressure applied to the films, FT-IR and polarization-modulated IRRAS spectroscopy developed in situ on the films indicated that all the peptides totally fold into intermolecular antiparallel beta-sheets. Calculated spectra of the amide region allowed us to define the orientation of the beta-strands compared to the interface. It is concluded that such beta-sheets remain flat-oriented without deep perturbation of zwitterionic phospholipids. Dansyl labelling at the N-terminus indicates that all the peptides are monomeric at a low concentration in aqueous buffer and bind to lipids with similar Dns burying. The affinities for zwitterionic lecithin mono- and bilayers, quantitatively estimated from buffer to lipid partition constants, monotonically increased with peptide length, indicating that hydrophobicity is a limiting parameter for lipid and membrane affinities. Peptides induced permeability increases on zwitterionic liposomes, they are strongly hemolytic towards human erythrocytes and their activity increases concurrently with length. Taking into account the lipid affinity, a hemolytic efficiency can be defined: at the same amount of peptide bound, this efficiency strongly increases with the peptide length. It is proposed that the first determinant step of membrane disturbance is the invasion of the outer membrane leaflet by these ideally amphipathic beta-sheeted structures lying flat at the interface, like large rafts depending on the number of beta-strands. (+info)
Exogenously incorporated ketocarotenoids in large unilamellar vesicles. Protective activity against peroxidation.
(38/927)
The ability of astaxanthin and canthaxanthin as chain-breaking antioxidants was studied in Cu(2+)-initiated peroxidation of phosphatidylcholine large unilamellar vesicles (LUVs). Both carotenoids increased the lag period that precedes the maximum rate of lipid peroxidation, though astaxanthin showed stronger activity. For these experiments, different amounts of xanthophylls were exogenously added to previously made LUVs, non-incorporated pigment being afterwards removed. Differential scanning calorimetry assays with L-beta,gamma-dimyristoyl-alpha-phosphatidylcholine LUVs demonstrated that xanthophylls incorporated as described interact with the lipid matrix becoming interspersed among the phospholipid molecules. (+info)
A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction.
(39/927)
The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37 degrees C for 1 hour, (125)I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas (125)I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the alpha-helix content of lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%) of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I Nichinan and normal r-proapoA-I showed similar particle size. To study cholesterol efflux, human skin fibroblasts were labeled with [(3)H]cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex. Fractional cholesterol efflux from [(3)H]cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation. Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I. Our data demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts. (+info)
Electric field effect on cholesterol-phospholipid complexes.
(40/927)
Monolayer mixtures of dihydrocholesterol and phospholipids at the air-water interface are used to model membranes containing cholesterol and phospholipids. Specific, stoichiometric interactions between cholesterol and some but not all phospholipids have been proposed to lead to the formation of condensed complexes. It is reported here that an externally applied electric field of the appropriate sign can destabilize these complexes, resulting in their dissociation. This is demonstrated through the application of an electric field gradient that leads to phase separations in otherwise homogeneous monolayers. This is observed only when the monolayer composition is close to the stoichiometry of the complex. The electric field effect is analyzed with the same mean field thermodynamic model as that used previously to account for pairs of upper miscibility critical points in these mixtures. The concentrations of dihydrocholesterol, phospholipid, and complex vary strongly and sometimes discontinuously in the monolayer membrane in the field gradient. The model is an approximation to a two-dimensional liquid in which molecules freely exchange between free and complexed form so that the chemical potentials are constant throughout the membrane. The calculations are illustrated for a complex of about 15 molecules, composed of 5 cholesterol molecules and 10 phospholipid molecules. (+info)