Computer simulation of small molecule permeation across a lipid bilayer: dependence on bilayer properties and solute volume, size, and cross-sectional area

Cell membrane permeation is required for most drugs to reach their biological target, and understanding this process is therefore crucial for rational drug design. Recent molecular dynamics simulations have studied the permeation of eight small molecules through a phospholipid bilayer. Unlike experiments, atomistic simulations allow the direct calculation of diffusion and partition coefficients of solutes at different depths inside a lipid membrane. Further analyses of the simulations suggest that solute diffusion is less size-dependent and solute partitioning more size-dependent than was commonly thought.     

Integral equation theories for predicting water structure around molecules

Water plays a crucial role in the structure and function of proteins and other biological macromolecules; thus, theories of aqueous solvation for these molecules are of great importance. However, water is a complex solvent whose properties are still not completely understood. Statistical mechanical integral equation theories predict the density distribution of water molecules around a solute so that all particles are fully represented and thus potentially both molecular and macroscopic properties are included. Here we discuss how several theoretical tools we have developed have been integrated into an integral equation theory designed for globular macromolecular solutes such as proteins. Our approach predicts the three-dimensional spatial and orientational distribution of water molecules around a solute. Beginning with a three-dimensional Ornstein-Zernike equation, a separation is made between a reference part dependent only on the spatial distribution of solvent and a perturbation part dependent also on the orientational distribution of solvent. The spatial part is treated at a molecular level by a modified hypernetted chain closure whereas the orientational part is treated as a Boltzmann prefactor using a quasi-continuum theory we developed for solvation of simple ions. A potential energy function for water molecules is also needed and the sticky dipole models of water, such as our recently developed soft-sticky dipole (SSD) model, are ideal for the proposed separation. Moreover, SSD water is as good as or better than three point models typically used for simulations of biological macromolecules in structural, dielectric and dynamics properties and yet is seven times faster in Monte Carlo and four times faster in molecular dynamics simulations. Since our integral equation theory accurately predicts results from Monte Carlo simulations for solvation of a variety of test cases from a single water or ion to ice-like clusters and ion pairs, the application of this theory to biological macromolecules is promising.     

Kinetic model for membrane transport. 1. Effects of membrane volume and partitioning kinetics

Equations for the transport of solutes through a membrane are derived, taking into account both the membrane volume and the partitioning kinetics, and have been found to involve two rate constants for solute transport, namely, those corresponding to solute transport from the solution to the membrane (k1) and from the membrane to the solution (k2). The time course followed before partitioning equilibrium has been attained, which is usually ignored, is shown to depend strongly on the relative magnitudes of k1 and k2.     

Static compression of articular cartilage can reduce solute diffusivity and partitioning: implications for the chondrocyte biological response.

Chondrocytes depend upon solute transport within the avascular extracellular matrix of adult articular cartilage for many of their biological activities. Alterations to bioactive solute transport may, therefore, represent a mechanism by which cartilage compression is transduced into cellular metabolic responses. We investigated the effects of cartilage static compression on diffusivity and partitioning of a range of model solutes including dextrans of molecular weights 3 and 40 kDa, and tetramethylrhodamine (a 430 Da fluorophore). New fluorescence methods were developed for real-time visualization and measurement of transport within compressed cartilage explants. Experimental design allowed for multiple measurements on individual explants at different compression levels in order to minimize confounding influences of compositional variations. Results demonstrate that physiological levels of static compression may significantly decrease solute diffusivity and partitioning in cartilage. Effects of compression were most dramatic for the relatively high molecular weight solutes. For 40 kDa dextran, diffusivity decreased significantly (p<0.01) between 8% and 23% compression, while partitioning of 3 and 40 kDa dextran decreased significantly (p<0.01) between free-swelling conditions and 8% compression. Since diffusivity and partitioning can influence pericellular concentrations of bioactive solutes, these observations support a role for perturbations to solute transport in mediating the cartilage biological response to compression.     

Effects of solute hydrophobicity and phospholipid composition on permeability of composite membranes containing phospholipids

Permeabilities of several solutes through the composite membranes containing phospholipids have been measured. They were inversely proportional to the content of the phospholipids in the membrane. Both the permeability of solutes and the degree of permeability change around the phase transition temperature of the phospholipids for the hydrophobic solutes such as n-butanol and salicylamide were larger than those for the hydrophilic solutes such as amino acids and pyridoxine. These results suggest thatthe permeation path of hydrophobic solutes is different from that of hydrophilic ones. The addition of phosphatidyl ethanolamine, phosphatidyl serine, or phosphatidic acid to the composite membrane influenced the solute permeability due to the introduced negative charge and/or the change in the molecular packing of phospholipid.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Direct enantiomeric separation of beta-blockers on ChyRoSine-A by supercritical fluid chromatography: supercritical carbon dioxide as transient in situ derivatizing agent.

The direct enantiomeric separation of a series of beta-blockers has been carried out on two chiral stationary phases (CSPs) derived from 3,5-dinitrobenzoyl tyrosine: the commercially available ChyRoSine-A and a recent improved version of this CSP. Using supercritical fluid chromatography (SFC), facile separations are achieved (1.1 less than Rs less than 7) within short analysis times. The parameters affecting the enantioselectivity (temperature, pressure, mobile phase nature, solute structure) have been investigated. The optimal mobile phase consists in a mixture of carbon dioxide-methanol-propylamine at 25 degrees C. The solute structure has a great influence on the enantioselectivity. For instance, both amine and hydroxyl protons are necessary for chiral discrimination to occur. Furthermore, the steroselectivity value is directly connected to the amine substituent steric bulkiness. Surprisingly, these solutes are poorly resolved using normal phase liquid chromatography (NPLC). Accordingly, the specific influence of carbon dioxide on the enantiomeric separation of 1,2-amino-alcohols have been investigated using various techniques such as nuclear magnetic resonance (NMR) or molecular modelisation. It has been shown that carbon dioxide acts as a complexing agent toward the amino-alcohol by setting up of a bridge with the hydroxyl and the amine protons of the solute. In that way, the resulting complex possesses lower acido-basic properties and a higher conformational rigidity, responsible for chiral discrimination.     

Experimental and computational studies of enantioseparation of structurally similar chiral compounds on amylose tris(3,5‐dimethylphenylcarbamate)

The enantioseparation of 14 structurally similar chiral solutes, with one or two chiral centers, are studied for a commercially important polysaccharide‐based chiral stationary phase, amylose tris(3,5‐dimethylphenylcarbamate) (ADMPC). Among these solutes, only two solutes show significant enantioresolutions of 2 to 2.5 in n‐hexane/2‐propanol (90/10, v/v) at 298 K. The retention factors of the chiral solutes vary significantly from 0.7 to 7.0, and they are compared with those of simpler nonchiral solutes having similar but fewer functional groups. The sorbent–solute H‐bonding interactions between the solute functional groups and the polymer C?O and NH functional groups are probed with attenuated total reflection infrared spectroscopy (ATR‐IR). The H‐bonding interactions of the polymer C?O and NH groups with the solutes result in changes in the IR amide band wavenumbers of ADMPC upon solute adsorption. The nanostructure of an ADMPC cavity and the potential interactions with the chiral solutes are proposed based on the sorbent–solute–solvent HPLC data, the sorbent–solute IR data, and the sorbent–solute molecular dynamics (MD) simulations. The results are consistent with the three point attachment hypothesis and indicate that a significant enantioresolution in ADMPC requires at least three different interaction sites for simultaneous H‐bonds and phenyl–phenyl interactions for phenylpropylamine (PPA) and various structurally similar chiral solutes. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Behaviour of small solutes and large drugs in a lipid bilayer from computer simulations

To reach their biological target, drugs have to cross cell membranes, and understanding passive membrane permeation is therefore crucial for rational drug design. Molecular dynamics simulations offer a powerful way of studying permeation at the single molecule level. Starting from a computer model proven to be able to reproduce the physical properties of a biological membrane, the behaviour of small solutes and large drugs in a lipid bilayer has been studied. Analysis of dihedral angles shows that a few nano seconds are sufficient for the simulations to converge towards common values for those angles, even if the starting structures belong to different conformations. Results clearly show that, despite their difference in size, small solutes and large drugs tend to lie parallel to the bilayer normal and that, when moving from water solution into biomembranes, permeants lose degrees of freedom. This explains the experimental observation that partitioning and permeation are highly affected by entropic effects and are size-dependent. Tilted orientations, however, occur when they make possible the formation of hydrogen bonds. This helps to understand the reason why hydrogen bonding possibilities are an important parameter in cruder approaches which predict drug absorption after administration. Interestingly, hydration is found to occur even in the membrane core, which is usually considered an almost hydrophobic region. Simulations suggest the possibility for highly polar compounds like acetic acid to cross biological membranes while hydrated. These simulations prove useful for drug design in rationalising experimental observations and predicting solute behaviour in biomembranes.     

Tuning membrane phase separation using nonlipid amphiphiles

Lipid phase separation may be a mechanism by which lipids participate in sorting membrane proteins and facilitate membrane-mediated biochemical signaling in cells. To provide new tools for membrane lipid phase manipulation that avoid direct effects on protein activity and lipid composition, we studied phase separation in binary and ternary lipid mixtures under the influence of three nonlipid amphiphiles, vitamin E (VE), Triton-X (TX)-100, and benzyl alcohol (BA). Mechanisms of additive-induced phase separation were elucidated using coarse-grained molecular dynamics simulations of these additives in a liquid bilayer made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC). From simulations, the additive's partitioning preference, changes in membrane thickness, and alterations in lipid order were quantified. Simulations showed that VE favored the DPPC phase but partitioned predominantly to the domain boundaries and lowered the tendency for domain formation, and therefore acted as a linactant. This simulated behavior was consistent with experimental observations in which VE promoted lipid mixing and dispersed domains in both gel/liquid and liquid-ordered/liquid-disordered systems. From simulation, BA partitioned predominantly to the DUPC phase, decreased lipid order there, and thinned the membrane. These actions explain why, experimentally, BA promoted phase separation in both binary and ternary lipid mixtures. In contrast, TX, a popular detergent used to isolate raft membranes in cells, exhibited equal preference for both phases, as demonstrated by simulations, but nonetheless, was a strong domain promoter in all lipid mixtures. Further analysis showed that TX increased membrane thickness of the DPPC phase to a greater extent than the DUPC phase and thus increased hydrophobic mismatch, which may explain experimental observation of phase separation in the presence of TX. In summary, these nonlipid amphiphiles provide new tools to tune domain formation in model vesicle systems and could provide the means to form or disperse membrane lipid domains in cells, in addition to the well-known methods involving cholesterol enrichment and sequestration.     

Nature of alcohol and anesthetic action on cooperative membrane equilibria.

A generalized, colligative thermodynamic framework is used to treat the action of solutes on cooperative membrane equilibria. Configurational entropy, the randomness imparted by solutes through the partitioning or mixing process, is implicated as the energetic driving force for the action of anesthetics on cooperative membrane equilibria. The equilibria predicted to be most sensitive to solute action--in which the dilute solute causes a perturbation equivalent to a large change in temperature--are (1) low-enthalpy processes that coincide with (2) large partitioning differences between states. The model stresses that solutes do not act at a single site, but on both states in an equilibrium, and that the perturbation is determined by the difference in entropy. Evidence for the thermodynamic framework is obtained from the partitioning behavior of the general anesthetic 1-hexanol into a model lecithin (DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine) membrane as a function of temperature and alcohol concentration. The low-enthalpy equilibrium between the gel (L beta') and ripple states (P beta') (pretransition) is more sensitive to 1-hexanol than the high-enthalpy equilibrium between the ripple (P beta') and fluid bilayer states (L alpha) (main transition). The perturbations of both equilibria are accurately described by the colligative thermodynamic framework. The results suggest that alcohols and anesthetics act through entropy to upset the natural thermal balance that maintains native membrane architecture.     

A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding.

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

Uptake and release protocol for assessing membrane binding and permeation by way of isothermal titration calorimetry

The activity of many biomolecules and drugs crucially depends on whether they bind to biological membranes and whether they translocate to the opposite lipid leaflet and trans aqueous compartment. A general strategy to measure membrane binding and permeation is the uptake and release assay, which compares two apparent equilibrium situations established either by the addition or by the extraction of the solute of interest. Only solutes that permeate the membrane sufficiently fast do not show any dependence on the history of sample preparation. This strategy can be pursued for virtually all membrane-binding solutes, using any method suitable for detecting binding. Here, we present in detail one example that is particularly well developed, namely the nonspecific membrane partitioning and flip-flop of small, nonionic solutes as characterized by isothermal titration calorimetry. A complete set of experiments, including all sample preparation procedures, can typically be accomplished within 2 days. Analogous protocols for studying charged solutes, virtually water-insoluble, hydrophobic compounds or specific ligands are also considered.     

Protein Partitioning into Ordered Membrane Domains: Insights from Simulations

Cellular membranes are laterally organized into domains of distinct structures and compositions by the differential interaction affinities between various membrane lipids and proteins. A prominent example of such structures are lipid rafts, which are ordered, tightly packed domains that have been widely implicated in cellular processes. The functionality of raft domains is driven by their selective recruitment of specific membrane proteins to regulate their interactions and functions; however, there have been few general insights into the factors that determine the partitioning of membrane proteins between coexisting liquid domains. In this work, we used extensive coarse-grained and atomistic molecular dynamics simulations, potential of mean force calculations, and conceptual models to describe the partitioning dynamics and energetics of a model transmembrane domain from the linker of activation of T cells. We find that partitioning between domains is determined by an interplay between protein-lipid interactions and differential lipid packing between raft and nonraft domains. Specifically, we show that partitioning into ordered domains is promoted by preferential interactions between peptides and ordered lipids, mediated in large part by modification of the peptides by saturated fatty acids (i.e., palmitoylation). Ordered phase affinity is also promoted by elastic effects, specifically hydrophobic matching between the membrane and the peptide. Conversely, ordered domain partitioning is disfavored by the tight molecular packing of the lipids therein. The balance of these dominant drivers determines partitioning. In the case of the wild-type linker of activation of T cells transmembrane domain, these factors combine to yield enrichment of the peptide at L_o/L_d interfaces. These results define some of the general principles governing protein partitioning between coexisting membrane domains and potentially explain previous disparities among experiments and simulations across model systems.     

Effect of the solute molecular structure on its enantioresolution on cellulose tris(3,5-dimethylphenylcarbamate)

The effects of the molecular structures for 13 structurally similar chiral solutes on their HPLC retention and enantioresolutions on a commercially important polysaccharide-based chiral stationary phase, cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) are studied. Among these 13 solutes, only methyl ephedrine (MEph) shows significant enantioresolution. The retention factors of these chiral solutes vary significantly from 0.7 to 3.2 in n-hexane/2-propanol (90/10, v/v) at 298 K. The retention factors of some simpler non-chiral solutes having similar but fewer functional groups than their chiral counterparts are also studied under the same conditions and are compared to those of the chiral solutes. The H-bonding interactions between the functional groups of the solute and the C=O and NH functional groups of the polymer are probed with attenuated total reflection-infrared spectroscopy (ATR-IR) for the polymer, for binary sorbent-solute systems. The CDMPC IR amide band wavenumbers change significantly, indicating H-bonding interactions of the polymer C=O and NH groups with the solutes. The elution orders predicted for the enantiomers of these chiral solutes using molecular dynamics (MD) simulations of the polymer-solute binary systems are consistent with the HPLC results. The CDMPC cavity nano-structure and the potential interactions with chiral solutes are proposed based on HPLC data, IR data, and the simulations. The results are consistent with the three-point attachment model and support the hypothesis that significant enantioresolution requires at least three different synergistic interactions which can be a combination of steric hindrance, H-bonding, or pi-pi interactions.     

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solute’s oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.     

Local Partition Coefficients Govern Solute Permeability of Cholesterol-Containing Membranes

The permeability of lipid membranes for metabolic molecules or drugs is routinely estimated from the solute’s oil/water partition coefficient. However, the molecular determinants that modulate the permeability in different lipid compositions have remained unclear. Here, we combine scanning electrochemical microscopy and molecular-dynamics simulations to study the effect of cholesterol on membrane permeability, because cholesterol is abundant in all animal membranes. The permeability of membranes from natural lipid mixtures to both hydrophilic and hydrophobic solutes monotonously decreases with cholesterol concentration [Chol]. The same is true for hydrophilic solutes and planar bilayers composed of dioleoyl-phosphatidylcholine or dioleoyl-phosphatidyl-ethanolamine. However, these synthetic lipids give rise to a bell-shaped dependence of membrane permeability on [Chol] for very hydrophobic solutes. The simulations indicate that cholesterol does not affect the diffusion constant inside the membrane. Instead, local partition coefficients at the lipid headgroups and at the lipid tails are modulated oppositely by cholesterol, explaining the experimental findings. Structurally, these modulations are induced by looser packing at the lipid headgroups and tighter packing at the tails upon the addition of cholesterol.