Thermodynamic characteristics of mixed monolayers of amphotericin B and cholesterol

Surface-pressure (Pi) and surface-area isotherms as a function of surface area were measured for monolayers of amphotericin B (AmB) and cholesterol mixtures at the air/water interface at 10, 20, and 30 degrees C. When chloroform/methanol was used as a spreading solvent, the Pi-A isotherms of the mixed monolayers exhibited characteristic transitions from the gas to liquid-expanded, then liquid-condensed, and finally the solid state. The expanding effect in monolayers was accompanied by a large Pi-A hysteresis and a positive excess of free energy of mixing at high Pi. At low Pi, a condensing effect was observed with the most significant deviation from ideality occurring at a mole fraction of AmB (XAmB) of 0.67. Free energy calculations revealed a condensing effect at low Pi and an expanding effect at high Pi except at 30 degrees C, where a condensing effect was observed for XAmB around 0.33. In contrast, when 2-propanol/water was used as spreading solvent, the mixed monolayers at 20 degrees C exhibited Pi-A isotherms which obey van der Waals equation of state, with no visible transitions, low hysteresis, a condensing effect, and a negative free energy of mixing. The most stable monolayers were produced from mixtures of AmB and cholesterol with a 2:1 stoichiometry.     

Large molecular assembly of amphotericin B formed in ergosterol-containing membrane evidenced by solid-state NMR of intramolecular bridged derivative

Amphotericin B (AmB 1) is known to assemble and form an ion channel across biomembranes. We have recently reported that conformation-restricted derivatives of AmB 2-4 show different ergosterol preferences in ion-channel assays, which suggested that the orientation of the mycosamine strongly affects the sterol selectivity of AmB. The data allowed us to assume that compound 3 showing the highest selectivity would reflect the active conformation of AmB in the channel assembly. In this study, to gain further insight into the active conformation of AmB, we prepared a new intramolecular-bridged derivative 5, where the linker encompassed a hydrophilic glycine moiety. The derivative has almost equivalent ion-channel activity to those of AmB and 3. The antifungal activity of 5 compared with 3 improves significantly, possibly because the increasing hydrophilicity in the linker enhances the penetrability through the fungal cell wall. Conformation of 5 was well converged and very similar to that of 3, thus further supporting the notion that the conformations of these derivatives reproduce the active structure of AmB in the channel complex. Then we used the derivative to probe the mobility of AmB in the membrane by solid-state NMR. To measure dipolar couplings and chemical shift anisotropies, we incorporated [1-(13)C,(15)N]glycine into the linker. The results indicate that 5 is mostly immobilized in ergosterol-containing DMPC bilayers, implying formation of large aggregates of 5. Meanwhile some fraction of 5 remains mobile in sterol-free DMPC bilayers, suggesting promotion of AmB aggregation by ergosterol.     

Differential effects of cholesterol, ergosterol and lanosterol on a dipalmitoyl phosphatidylcholine membrane: a molecular dynamics simulation study

Lipid raft/domain formation may arise as a result of the effects of specific sterols on the physical properties of membranes. Here, using molecular dynamics simulation, we examine the effects of three closely-related sterols, ergosterol, cholesterol, and lanosterol, at a biologically relevant concentration (40 mol %) on the structural properties of a model dipalmitoyl phosphatidylcholine (DPPC) membrane at 309 and 323 K. All three sterols are found to order the DPPC acyl tails and condense the membrane relative to the DPPC liquid-phase membrane, but each one does this to a significantly different degree. The smooth alpha-face of ergosterol, together with the presence of tail unsaturation in this sterol, leads to closer interaction of ergosterol with the lipids and closer packing of the lipids with each other, so ergosterol has a higher condensing effect on the membrane, as reflected by the area per lipid. Moreover, ergosterol induces a higher proportion of trans lipid conformers, a thicker membrane, and higher lipid order parameters and is aligned more closely with the membrane normal. Ergosterol also positions itself closer to the bilayer/water interface. In contrast, the rough alpha-face of lanosterol leads to a less close interaction of the steroid ring system with the phospholipid acyl chains, and so lanosterol orders, straightens, and packs the lipid acyl chains less well and is less closely aligned with the membrane normal. Furthermore, lanosterol lies closer to the relatively disordered membrane center than do the other sterols. The behavior of cholesterol in all the above respects is intermediate between that of lanosterol and ergosterol. The findings here may explain why ergosterol is the most efficient of the three sterols at promoting the liquid-ordered phase and lipid domain formation and may also furnish part of the explanation as to why cholesterol is evolutionarily preferred over lanosterol in higher-vertebrate plasma membranes.     

Solvation of transmembrane proteins by isotropic membrane mimetics: a molecular dynamics study

Mixtures of organic solvents are often used as membrane mimetics in structure determination of transmembrane proteins by solution NMR; however, the mechanism through which these isotropic solvents mimic the anisotropic environment of cell membranes is not known. Here, we use molecular dynamics simulations to study the solvation thermodynamics of the c-subunit of Escherichia coli F1F0 ATP synthase in membrane mimetic mixtures of methanol, chloroform, and water with varying fractions of components as well as in lipid bilayers. We show that the protein induces a local phase separation of the solvent components into hydrophobic and hydrophilic layers, which provides the anisotropic solvation environment to stabilize the amphiphilic peptide. The extent of this effect varies with solvent composition and is most pronounced in the ternary methanol-chloroform-water mixtures. Analysis of the solvent structure, including the local mole fraction, density profiles, and pair distribution functions, reveals considerable variation among solvent mixtures in the solvation environment surrounding the hydrophobic transmembrane region of the protein. Hydrogen bond analysis indicates that this is primarily driven by the hydrogen-bonding propensity of the essential Asp(61) residue. The impact of the latter on the conformational stability of the solvated protein is discussed. Comparison with the simulations in explicit all-atom models of lipid bilayer indicates a higher flexibility and reduced structural integrity of the membrane mimetic solvated c-subunit. This was particularly true for the deprotonated form of the protein and found to be linked to solvent stabilization of the charged Asp(61).     

Reference interaction site model and molecular dynamics study of structure and thermodynamics of methanol

Thermodynamic and structural properties of various models of liquid methanol are investigated in the framework provided by the reference interaction site model (RISM) theory of molecular fluids. The theoretical predictions are systematically compared with molecular dynamics simulations both at ambient conditions and along a few supercritical isotherms. RISM results for the liquid-vapor phase separation are also obtained and assessed against available Gibbs ensemble Monte Carlo data. At ambient conditions, the theoretical correlations weakly depend on the specific details of the molecular models and reproduce the simulation results with different degrees of accuracy, depending on the pair of interaction sites considered. The position and the strength of the hydrogen bond are quite satisfactorily predicted. RISM results for the internal energy are almost quantitative whereas the pressure is generally overestimated. As for the liquid-vapor phase coexistence, RISM predictions for the vapor branch and for the critical temperature are quite accurate; on the other side, the liquid branch densities, and consequently the critical density, are underestimated. We discuss our results in terms of intrinsic limitations, and suitable improvements, of the RISM approach in describing the physical properties of polar fluids, and in the perspective of a more general investigation of mixtures of methanol with nonpolar fluids of specific interest in the physics of associating fluids.     

Pore-spanning lipid membrane under indentation by a probe tip: a molecular dynamics simulation study

We study the indentation of a free-standing lipid membrane suspended over a nanopore on a hydrophobic substrate by means of molecular dynamics simulations. We find that in the course of indentation the membrane bends at the point of contact and the fringes of the membrane glide downward intermittently along the pore edges and stop gliding when the fringes reach the edge bottoms. The bending continues afterward, and the large strain eventually induces a phase transition in the membrane, transformed from a bilayered structure to an interdigitated structure. The membrane is finally ruptured when the indentation goes deep enough. Several local physical quantities in the pore regions are calculated, which include the tilt angle of lipid molecules, the nematic order, the included angle, and the distance between neighboring lipids. The variations of these quantities reveal many detailed, not-yet-specified local structural transitions of lipid molecules under indentation. The force-indentation curve is also studied and discussed. The results make a connection between the microscopic structure and the macroscopic properties and provide deep insight into the understanding of the stability of a lipid membrane spanning over nanopore.     

Lattice dynamics and interaction potentials in molecular crystals

This paper, based essentially on the work done in recent years in our laboratory, presents a critical analysis of harmonic and anharmonic calculations of crystal vibrations in the determination of intermolecular potentials. The main purpose is to show that the dynamical properties are specifically sensitive to different terms of the potential and thus that information extracted from vibrational spectra of crystals is of the greatest importance for the theory of intermolecular forces. The most important conclusion is that these calculations point to the development of anisotropic short-range potentials and orient future researches towards the more complex and elaborate anharmonic treatment of crystal vibrations.     

The interaction of water with MOF-5 simulated by molecular dynamics

Force field parameters for use with metal-organic framework-5 (MOF-5 or IRMOF-1) are presented. Flexibility within the framework is included in this model, so that structural changes upon interaction with adsorbate molecules can be observed and quantified. The model was validated by comparing simulated lattice parameters of pure MOF-5 with X-ray diffraction results. For the first time, molecular dynamics simulations have been performed that show how water interacts with MOF-5. The framework is stable at water contents up to 2.3% by mass, but distortion in the lattice structure is already evident. At water contents of 3.9% and higher, the framework collapses because of the replacement of MOF O atoms by water O atoms in the Zn coordination shells. As a result, inorganic MOF O atoms are no longer coordinated by four Zn ions, and benzene dicarboxylate linkers are no longer tethered to Zn centers.     

Ab initio molecular dynamics study of manganese porphine hydration and interaction with nitric oxide

The authors use ab initio molecular dynamics and the density functional theory+U (DFT+U) method to compute the hydration environment of the manganese ion in manganese (II) and manganese (III) porphines (MnP) dispersed in liquid water. These are intended as simple models for more complex water soluble porphyrins, which have important physiological and electrochemical applications. The manganese ion in Mn(II)P exhibits significant out-of-porphine plane displacement and binds strongly to a single H2O molecule in liquid water. The Mn in Mn(III)P is on average coplanar with the porphine plane and forms a stable complex with two H2O molecules. The residence times of these water molecules exceed 15 ps. The DFT+U method correctly predicts that water displaces NO from Mn(III)P-NO, but yields an ambiguous spin state for the MnP(II)-NO complex.     

Energetics of the human Tel-22 quadruplex-telomestatin interaction: a molecular dynamics study

The formation and stabilization of telomeric quadruplexes has been shown to inhibit the activity of telomerase, thus establishing telomeric DNA quadruplex as an attractive target for cancer therapeutic intervention. In this context, telomestatin, a G-quadruplex-specific ligand known to bind and stabilize G-quadruplex, is of great interest. Knowledge of the three-dimensional structure of telomeric quadruplex and its complex with telomestatin in solution is a prerequisite for structure-based rational drug design. Here, we report the relative stabilities of human telomeric quadruplex (AG3[T2AG3]3) structures under K+ ion conditions and their binding interaction with telomestatin, as determined by molecular dynamics simulations followed by energy calculations. The energetics study shows that, in the presence of K+ ions, mixed hybrid-type Tel-22 quadruplex conformations are more stable than other conformations. The binding free energy for quadruplex-telomestatin interactions suggests that 1:2 binding is favored over 1:1 binding. To further substantiate our results, we also calculated the change in solvent-accessible surface area (DeltaSASA) and heat capacity (DeltaCp) associated with 1:1 and 1:2 binding modes. The extensive investigation performed for quadruplex-telomestatin interaction will assist in understanding the parameters influencing the quadruplex-ligand interaction and will serve as a platform for rational drug design.     

Nonspecific interaction forces at water-membrane interface by forced molecular dynamics simulations

Nonspecific interactions are the main driving forces for the behavior of molecules with great affinity for biologic membranes. To investigate not only the molecular details of these interactions but to estimate their magnitude as well, the theoretical method of Forced Molecular Dynamics Simulations, based on the Atomic Force Spectroscopy experimental technique, was applied. In this approach, an additional one-dimensional elastic force, representing the cantilever probe, was incorporated to the force field of a Molecular Dynamics computational program. This force represents a spring fixed on one end to a selected atom of the molecule; the other end of the spring is displaced at constant velocity to pull the molecule out of the membrane. The force experimented by the molecule due to the spring, is proportional to the spring elongation relative to its equilibrium position. This value is registered during the entire simulation, and its maximum value will determine the molecule-membrane interaction force. Nonexplicit medium simulations were carried out. Polar and apolar media were considered according to their polarizability degree and a specific dielectric constant value was assigned. In this approach, the membrane was considered as the apolar region limited by two flat surfaces with a polar aqueous medium. The potential energy discontinuity at the interfaces was smoothed by considering the polarization-induced effects using the image method. The results of this methodology are presented using a small system, a single Alanine amino acid model, which enables extended simulations in a microsecond time scale. The confinement of this amino acid at the interface reduces its degrees of freedom and forces it to adopt one of the six defined conformations. A correlation between these stable structures at the water-membrane interface and the interaction force value was determined.     

Role of cholesterol and polyunsaturated chains in lipid-protein interactions: molecular dynamics simulation of rhodopsin in a realistic membrane environment

We present a 118-ns molecular dynamics simulation of rhodopsin embedded in a bilayer composed of a 2:2:1 mixture of 1-stearoyl-2-docosahexaenoyl-phosphatidylcholine (SDPC), 1-stearoyl-2-docosahexaenoyl-phosphatidylethanolamine (SDPE), and cholesterol, respectively. The simulation demonstrates that the protein breaks the lateral and transverse symmetry of the bilayer. Lipids near the protein preferentially reorient such that their unsaturated chains interact with the protein, while the distribution of cholesterol in the membrane complements the variations in rhodopsin's transverse profile. The latter phenomenon suggests a molecular-level mechanism for the experimental finding that cholesterol stabilizes the native dark-adapted state of rhodopsin without binding directly to the protein.     

Molecular dynamics studies of the molecular structure and interactions of cholesterol superlattices and random domains in an unsaturated phosphatidylcholine bilayer membrane

The effect of the molecular organization of lipid components on the properties of the bilayer membrane has been a topic of increasing interest. Several experimental and theoretical studies have suggested that cholesterol is not randomly distributed in the fluid-state lipid bilayer but forms nanoscale domains. Several cholesterol-enriched nanodomain structures have been proposed, including rafts, regular or maze arrays, complexes, and superlattices. At present, the molecular mechanisms by which lipid composition influences the formation and stability of lipid nanodomains remain unclear. In this study, we have used molecular dynamics (MD) simulations to investigate the effects of the molecular organization of cholesterol--superlattice versus random--on the structure of and interactions between lipids and water in lipid bilayers of cholesterol and 1-palmitoyl-2-oleoylphosphatidylcholine (cholesterol/POPC) at a fixed cholesterol mole fraction of 0.40. On the basis of four independent replicates of 200-ns MD simulations for a superlattice or random bilayer, statistically significant differences were observed in the lipid structural parameters, area per lipid, density profile, and acyl chain order profile, as well as the hydrogen bonding between various pairs (POPC and water, cholesterol and water, and POPC and cholesterol). The time evolution of the radial distribution of the cholesterol hydroxy oxygen suggests that the lateral distribution of cholesterol in the superlattice bilayer is more stable than that in the random bilayer. Furthermore, the results indicate that a relatively long simulation time, more than 100 ns, is required for these two-component bilayers to reach equilibrium and that this time is influenced by the initial lateral distribution of lipid components.     

Theoretical study of intramolecular interaction energies during dynamics simulations of oligopeptides by the fragment molecular orbital-Hamiltonian algorithm method

We analyzed the interaction energies between residues (fragments) in an oligopeptide occurring during dynamic simulations by using the fragment molecular orbital-Hamiltonian algorithm (FMO-HA) method, an ab initio MO-molecular dynamics technique. The FMO method enables not only calculation of large molecules based on ab initio MO but also easy evaluation of interfragment interaction energies. The glycine pentamer [(Gly)(5)] and decamer [(Gly)(10)] were divided into five and ten fragments, respectively. alpha-helix structures of (Gly)(5) and (Gly)(10) were stabilized by attractive interaction energies owing to intramolecular hydrogen bonds between fragments n and n+3 (and n+4), and beta-strand structures were characterized by repulsive interaction energies between fragments n and n+2. We analyzed interfragment interaction energies during dynamics simulations as the peptides' geometries changed from alpha helix to beta strand. Intramolecular hydrogen bonds between fragments 2-4 and 2-5 control the geometrical preference of (Gly)(5) for the beta-strand structure. The pitch of one turn of the alpha helix of (Gly)(10) elongated and thus weakened during dynamics due to a shifting of the intramolecular hydrogen bonds, and enabled the beta-strand structure to form. Changes in interaction energies due to the intramolecular hydrogen bonds controlled the tendency toward alpha-helix or beta-strand structure of (Gly)(5) and (Gly)(10). Evaluation of interfragment interaction energies during dynamics simulations thus enabled detailed analysis of the process of the geometrical changes occurring in oligopeptides.     

A fourier transform infrared spectroscopic study of the molecular interaction in synthetic bilayer membrane

Fourier transform infrared spectroscopy is used to study the molecular interaction between gramicidin D and bilayer membranes, dioctadecadimethylammonium bromide (2C₁₈N⁺2C₁Br⁻), and 1,2-di-palmytoyl- -α-phosphatidylcholine (DPPC). Frequencies and bandwidths of the symmetric CH₂ stretching band measured as a function of temperature are used to study variation of packing of alkyl chains. The bilayer membrane prepared from 2C₁₈N⁺2C₁Br⁻ is found to have a gel to liquid crystal phase transition at 42°C. The presence of gramicidin in the membrane causes an increase in the mobility of the alkyl chain and also a decrease in the abruptness of the transition.The frequencies of amide I and II bands of gramicidin reflecting secondary structures of polypeptides are used to identify its conformation in membranes and to study the interaction between gramicidin and the matrices. Gramicidin is found to have hydrophobic interaction with 2C₁₈N⁺2C₁Br⁻, whereas it has both hydrophobic and hydrophilic interactions with DPPC.     

Study of penetration of amphotericin B into cholesterol or ergosterol containing dipalmitoyl phosphatidylcholine Langmuir monolayers

The interactions of amphotericin B (AmB) with sterols and phospholipids have been studied by adsorption of AmB from aqueous solutions into Langmuir monolayers from dipalmitoyl phosphatidylcholine (DPPC), ergosterol, cholesterol and their mixtures. The results show that AmB exhibits stronger interaction with cholesterol than ergosterol in one-component monolayers. However, for DPPC–sterol monolayers, the effectiveness of AmB penetration depends on the proportion of both film components in the mixed film as well as on the strength of interaction between DPPC and particular sterol.     

Study of the interaction between progesterone and beta-cyclodextrin by electrochemical techniques and steered molecular dynamics

The interaction of progesterone with beta-cyclodextrin (beta-CD) was studied by differential pulse polarography. The aim of the present work was to study the effect of beta-CD on the electrochemical behavior of progesterone in aqueous solution and also to analyze the molecular interactions involved in formation of the inclusion complex. The complex with stoichiometry of 1:1 was thermodynamically characterized. In addition, steered molecular dynamics (SMD) was used to investigate the energetic properties of formation of the inclusion complex along four different pathways (reaction coordinates), considering two possible orientations. From multiple trajectories along these pathways, the potentials of mean force for formation of the beta-CD progesterone inclusion complex were calculated. The energy analysis was in good agreement with the experimental results. In the beta-CD progesterone inclusion complex, a large portion of the steroid skeleton is included in the beta-CD cavity. The lowest energy was found when the D-ring of the guest molecule is located near the secondary hydroxyls of the beta-CD cavity. In the most probable orientation, one intermolecular hydrogen bond is formed between the O of the C-20 keto group of the progesterone and a secondary hydroxyl of the beta-CD.     

Interaction of amphotericin B and its selected derivatives with membranes: molecular modeling studies

Amphotericin B (AmB) is a well-known antifungal antibiotic that has been used in the clinic for about five decades. Despite its chemotherapeutic importance, AmB is quite toxic and many efforts have been made to improve its pharmacological properties, e.g., by chemical modifications. The lipid membrane is a molecular target for AmB, however, due to heterogeneity of its components, the molecular mechanism of AmB action is still unclear. The lack of this knowledge hinders rational designing of new and less toxic AmB derivatives. Our review is a critical presentation of the current understanding of AmB molecular mechanism of action at the membrane level. Except the experimental approach, the extensive overview of molecular modeling studies, performed mostly in our lab, is presented. The results of interactions between AmB or some of its derivatives and lipid model membranes are discussed. In our studies, different biomembrane models and different associate states of the antibiotic were included. Presented molecular modeling approach is especially valuable with regard to a new paradigm of the structure of lipid membrane containing liquid-ordered domains. Hopefully, all these complementary experimental/computational approaches are going to reach the point at which a new hypothesis about molecular mechanism of AmB activity and selectivity will be put forward.