The Effects of Calcium on Lipid-Protein Interactions and Ion Flux in the Cx26 Connexon Embedded into a POPC Bilayer.

Gap junctions provide a communication pathway between adjacent cells. They are formed by paired connexons that reside in the plasma membrane of their respective cell and their activity can be modulated by the bilayer composition. In this work, we study the dynamic behavior of a Cx26 connexon embedded in a POPC lipid bilayer, studying: the membrane protein interactions and the ion flux though the connexon pore. We analyzed extensive atomistic molecular dynamics simulations for different conditions, with and without calcium ions. We found that lipid-protein interactions were mainly mediated by hydrogen bonds. Specific amino acids were identified forming hydrogen bonds with the POPC lipids (ARG98, ARG127, ARG165, ARG216, LYS22, LYS221, LYS223, LYS224, SER19, SER131, SER162, SER219, SER222, THR18 and TYR97, TYR155, TYR212, and TYR217). In the presence of calcium ions, we found subtle differences on the HB lifetimes. Finally, these MD simulations are able to identify and explain differential chlorine flux through the pore depending on the presence or absence of the calcium ions and its distribution within the pore.

Electropore Formation in Mechanically Constrained Phospholipid Bilayers.

Molecular dynamics simulations of lipid bilayers in aqueous systems reveal how an applied electric field stabilizes the reorganization of the water-membrane interface into water-filled, membrane-spanning, conductive pores with a symmetric, toroidal geometry. The pore formation process and the resulting symmetric structures are consistent with other mathematical approaches such as continuum models formulated to describe the electroporation process. Some experimental data suggest, however, that the shape of lipid electropores in living cell membranes may be asymmetric. We describe here the axially asymmetric pores that form when mechanical constraints are applied to selected phospholipid atoms. Electropore formation proceeds even with severe constraints in place, but pore shape and pore formation time are affected. Since lateral and transverse movement of phospholipids may be restricted in cell membranes by covalent attachments to or non-covalent associations with other components of the membrane or to membrane-proximate intracellular or extracellular biomolecular assemblies, these lipid-constrained molecular models point the way to more realistic representations of cell membranes in electric fields.

Size-controlled nanopores in lipid membranes with stabilizing electric fields.

Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes under the influence of an applied electric field. However, the study of the structure and transport properties of electropores by means of MD has been hampered by difficulties in the maintenance of a stable electropore in the typically small simulated membrane patches. We describe a new simulation scheme in which an initially larger porating field is systematically reduced after pore formation to lower stabilizing values to produce stable, size-controlled electropores, which can then be characterized at the molecular level. A new method allows the three-dimensional modeling of the irregular shape of the pores obtained as well as the quantification of its volume. The size of the pore is a function of the value of the stabilizing field. At lower fields the pore disappears and the membrane recovers its normal shape, although in some cases long-lived, fragmented pores containing unusual lipid orientations in the bilayer are observed.

Effects of dimethyl sulfoxide on lipid membrane electroporation.

Pores can be generated in lipid membranes by the application of an external electric field or by the addition of particular chemicals such as dimethyl sulfoxide (DMSO). Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes in both situations. By means of MD simulations, we address the formation of electropores in cholesterol-containing lipid bilayers under the influence of DMSO. We show how a combination of physical and chemical mechanisms leads to more favorable conditions for generating membrane pores and, in particular, how the addition of DMSO to the medium significantly reduces the minimum electric field required to electroporate a lipid membrane. The strong alteration of membrane transversal properties and the energetic stabilization of the hydrophobic pore stage by DMSO provide the physicochemical mechanisms that explain this effect.

Effects of dimethyl sulfoxide in cholesterol-containing lipid membranes: a comparative study of experiments in silico and with cells.

Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca(2+)) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations.

Structural and kinetic molecular dynamics study of electroporation in cholesterol-containing bilayers.

We present a numerical study of pore formation in lipid bilayers containing cholesterol (Chol) and subjected to a transverse electric field. Molecular dynamics simulations of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DOPC) membranes reveal the formation of a pore when an electric field of 325 mV/nm is applied. The minimum electric field needed for membrane permeabilization strongly increases with the addition of cholesterol above 10 mol %, reaching 750 mV/nm for 40 mol % Chol. Analysis of simulations of DOPC/Chol bilayers suggests this is caused by a substantial increment of membrane cohesion. Simulations also show that pore formation kinetics is much slower at high Chol contents.

Theoretical insight into the hydroxylamine oxidoreductase mechanism.

The multiheme enzyme hydroxylamine oxidoreductase from the autotrophic bacteria Nitrosomonas europaea catalyzes the conversion of hydroxylamine to nitrite, with a complicate arrangement of heme groups in three subunits. As a distinctive feature, the protein has a covalent linkage between a tyrosyl residue of one subunit and a meso carbon atom of the heme active site of another. We studied the influence of this bond in the catalysis from a theoretical perspective through electronic structure calculations at the density functional theory level, starting from the crystal structure of the protein. Geometry optimizations of proposed reaction intermediates were used to calculate the dissociation energy of different nitrogen containing ligands, considering the presence and absence of the meso tyrosyl residue. The results indicate that the tyrosine residue enhances the binding of hydroxylamine, and increases the stability of a Fe(III)NO intermediate, while behaving indifferently in the Fe(II)NO form. The calculations performed on model systems including neighboring aminoacids revealed the probable formation of a bidentate hydrogen bond between the Fe(III)H(2)O complex and Asp 257, in a high-spin aquo complex as the resting state. Characterization of non-planar heme distortions showed that the meso-substituent induces significant ruffling in the evaluated intermediates.

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study.

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 x Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe(+2)) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe-S bond and yields five coordinate complexes. In the ferric (Fe(+3)) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe(+3)-NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe(+2)-NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.

Protective effect of prolactin and placental lactogen on NO-induced Nb2 lymphoma cell apoptosis.

Nitric oxide (NO) is an important modulator involved in immune regulation. Here, we describe conditions under which NO-donors induce apoptosis on Nb2 lymphoma cells, as evidenced by decreased cell viability and increased hypodiploid DNA content determined by flow cytometry. In addition, DNA fragmentation typical of apoptosis was shown by agarose gel electrophoresis. This apoptosis was accompanied by a significant increase of caspase-3-like enzymatic activity. Both ovine prolactin (oPRL) and ovine placental lactogen (oPL) exerted a protective effect on the NO-donor-induced apoptosis. Furthermore, dexamethasone (Dex)-induced cell death was also associated with caspase-3-like activity and oPL had the same potency as oPRL in its protective effect on Dex-induced apoptosis of Nb2 cells.