Effect of "helper lipid" on lipoplex electrostatics

Lipoplexes, which are complexes between cationic liposomes (L+) and nucleic acids, are commonly used as a nucleic acid delivery system in vitro and in vivo. This study aimed to better characterize cationic liposome and lipoplex electrostatics, which seems to play a major role in the formation and the performance of lipoplexes in vitro and in vivo. We characterized lipoplexes based on two commonly used monocationic lipids, DOTAP and DMRIE, and one polycationic lipid, DOSPA--each with and without helper lipid (cholesterol or DOPE). Electrical surface potential (Psi0) and surface pH were determined using several surface pH-sensitive fluorophores attached either to a one-chain lipid (4-heptadecyl hydroxycoumarin (C17HC)) or to the primary amino group of the two-chain lipids (1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-carboxyfluorescein (CFPE) and 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-N-7-hydroxycoumarin) (HC-DOPE). Zeta potentials of the DOTAP-based cationic liposomes and lipoplexes were compared with Psi0 determined using C17HC. The location and relatively low sensitivity of fluorescein to pH changes explains why CFPE is the least efficient in quantifying the differences between the various cationic liposomes and lipoplexes used in this study. The fact that, for all cationic liposomes studied, those containing DOPE as helper lipid have the least positive Psi0 indicates neutralization of the cationic charge by the negatively-charged phosphodiester of the DOPE. Zeta potential is much less positively charged than Psi0 determined by C17HC. The electrostatics affects size changes that occurred to the cationic liposomes upon lipoplex formation. The largest size increase (based on static light scattering measurements) for all formulations occurred at DNA-/L+ charge ratios 0.5-1. Comparing the use of the one-chain C17HC and the two-chain HC-DOPE for monitoring lipoplex electrostatics reveals that both are suitable, as long as there is no serum (or other lipidic assemblies) present in the medium; in the latter case, only the two-chain HC-DOPE gives reliable results. Increasing NaCl concentrations decrease surface potential. Neutralization by DNA is reduced in a NaCl-concentration-dependent manner.     

Atomic-scale structure and electrostatics of anionic palmitoyloleoylphosphatidylglycerol lipid bilayers with Na+ counterions

Anionic palmitoyloleoylphosphatidylglycerol (POPG) is one of the most abundant lipids in nature, yet its atomic-scale properties have not received significant attention. Here we report extensive 150-ns molecular dynamics simulations of a pure POPG lipid membrane with sodium counterions. It turns out that the average area per lipid of the POPG bilayer under physiological conditions is approximately 19% smaller than that of a bilayer built from its zwitterionic phosphatidylcholine analog, palmitoyloleoylphosphatidylcholine. This suggests that there are strong attractive interactions between anionic POPG lipids, which overcome the electrostatic repulsion between negative charges of PG headgroups. We demonstrate that interlipid counterion bridges and strong intra- and intermolecular hydrogen bonding play a key role in this seemingly counterintuitive behavior. In particular, the substantial strength and stability of ion-mediated binding between anionic lipid headgroups leads to complexation of PG molecules and ions and formation of large PG-ion clusters that act in a concerted manner. The ion-mediated binding seems to provide a possible molecular-level explanation for the low permeability of PG-containing bacterial membranes to organic solvents: highly polar interactions at the water/membrane interface are able to create a high free energy barrier for hydrophobic molecules such as benzene.     

Orientational polarisability of lipid membrane surfaces

Here we present a fluorescence method based on the Stokes shift of the voltage-sensitive dye di-8-ANEPPS to quantify the orientational polarisability of lipid membrane surfaces, i.e. the polarisability due to molecular reorientation. Di-8-ANEPPS is already an established probe of membrane dipole potential. Its use, therefore, as a probe of both the dipole potential and orientational polarisability allows a direct comparison of these two properties in an identical region of the lipid bilayer. We applied the new technique on phosphatidylcholine vesicles to study the effects of different degrees of hydrocarbon saturation and of the incorporation of cholesterol and some of its oxidized derivatives. We found that lipids with unsaturated chains had a lower orientational polarisability than those with saturated chains. This could be explained by a reduction in membrane dipole potential as a result of a decrease in lipid packing density. Cholesterol derivatives were found to either increase or decrease the orientational polarisability depending on their molecular structure. The varying effects could be explained by antagonistic effects of the dipole potential and membrane order, which are both changed to varying degrees by the cholesterol derivatives and which lead to increases and decreases in orientational polarisability, respectively.     

Orientational polarisability of lipid membrane surfaces

Here we present a fluorescence method based on the Stokes shift of the voltage-sensitive dye di-8-ANEPPS to quantify the orientational polarisability of lipid membrane surfaces, i.e. the polarisability due to molecular reorientation. Di-8-ANEPPS is already an established probe of membrane dipole potential. Its use, therefore, as a probe of both the dipole potential and orientational polarisability allows a direct comparison of these two properties in an identical region of the lipid bilayer. We applied the new technique on phosphatidylcholine vesicles to study the effects of different degrees of hydrocarbon saturation and of the incorporation of cholesterol and some of its oxidized derivatives. We found that lipids with unsaturated chains had a lower orientational polarisability than those with saturated chains. This could be explained by a reduction in membrane dipole potential as a result of a decrease in lipid packing density. Cholesterol derivatives were found to either increase or decrease the orientational polarisability depending on their molecular structure. The varying effects could be explained by antagonistic effects of the dipole potential and membrane order, which are both changed to varying degrees by the cholesterol derivatives and which lead to increases and decreases in orientational polarisability, respectively.     

Membrane electrostatics

In conclusion, charged membrane together with their adjacent electrolyte solution form a thermodynamic and physico-chemical entity. Their surfaces represent an exceptionally complicated interfacial system owing to intrinsic membrane complexity, as well as to the polarity and often large thickness of the interfacial region. Despite this, charged membranes can be described reasonably accurately within the framework of available theoretical models, provided that the latter are chosen on the basis of suitable criteria, which are briefly discussed in Section A. Interion correlations are likely to be important for the regular and/or rigid, thin membrane-solution interfaces. Lateral distribution of the structural membrane charge is seldom and charge distribution perpendicular to the membranes is nearly always electrostatically important. So is the interfacial hydration, which to a large extent determines the properties of the innermost part of the interfacial region, with a thickness of 2-3 nm. Fine structure of the ion double-layer and the interfacial smearing of the structural membrane charge decrease whilst the surface hydration increases the calculated value of the electrostatic membrane potential relative to the result of common Gouy-Chapman approximation. In some cases these effects partly cancel-out; simple electrostatic models are then fairly accurate. Notwithstanding this, it is at present difficult to draw detailed molecular conclusions from a large part of the published data, mainly owing to the lack of really stringent controls or calibrations. Ion binding to the membrane surface is a complicated process which involves charge-charge as well as charge-solvent interactions. Its efficiency normally increases with the ion valency and with the membrane charge density, but it is also strongly dependent on the physico-chemical and thermodynamic state of the membrane. Except in the case of the stereospecific ion binding to a membrane, the relatively easily accessible phosphate and carboxylic groups on lipids and integral membrane proteins are the main cation binding sites. Anions bind preferentially to the amine groups, even on zwitterionic molecules. Membrane structure is apt to change upon ion binding but not always in the same direction: membranes with bound ions can either expand or become more condensed, depending on the final hydrophilicity (polarity) of the membrane surface. The more polar membranes, as a rule, are less tightly packed and more fluid. Diffusive ion flow across a membrane depends on the transmembrane potential and concentration gradients, but also on the coulombic and hydration potentials at the membrane surface.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of electrostatics in protein-membrane interactions

Many experimental, structural and computational studies have established the importance of nonspecific electrostatics as a driving force for peripheral membrane association. Here we focus on this component of protein/membrane interactions by using examples ranging from phosphoinositide signaling to retroviral assembly. We stress the utility of the collaboration of experiment and theory in identifying and quantifying the role of electrostatics not only in contributing to membrane association, but also in affecting subcellular targeting, in the control of membrane binding, and in the organization of proteins and lipids at membrane surfaces.     

Mixed bilayer containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine: lipid complexation, ion binding, and electrostatics

Two mixed bilayers containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine at a ratio of 5:1 are simulated in NaCl electrolyte solutions of different concentration using the molecular dynamics technique. Direct NH.O and CH.O hydrogen bonding between lipids was observed to serve as the basis of interlipid complexation. It is deduced from our results and previous studies that dipalmitoylphosphatidylcholine alone is less likely to form interlipid complexes than in the presence of bound ions or other bilayer "impurities" such as dipalmitoylphosphatidylserine. The binding of counterions is observed and quantitated. Based upon the calculated ion binding constants, the Gouy-Chapman surface potential (theta) is calculated. In addition we calculated the electrostatic potential profile (Phi) by twice integrating the system charge distribution. A large discrepancy between and the value of Phi at the membrane surface is observed. However, at "larger" distance from the bilayer surface, a qualitative similarity in the z-profiles of Phi and psi(GC) is seen. The discrepancy between the two potential profiles near the bilayer surface is attributed to the discrete and nonbulk-like nature of water in the interfacial region and to the complex geometry of this region.     

Enhancement of phototransduction protein interactions by lipid surfaces

The G protein cascade of vision depends on two peripheral membrane proteins: the G protein, transducin (G(t)), and cGMP phosphodiesterase (PDE). Each has covalently attached lipids, and interacts with transduction components on the membrane surface. We have found that their surface interactions are critically dependent on the nature of the lipid. Membranes enhance their protein-protein interactions, especially if electrostatic attraction is introduced with positively charged lipids. These interactions are less enhanced on highly curved surfaces, but are most enhanced by unsaturated or bulky acyl chains. On positively charged membranes, G(t) assembles at a high enough density to form two-dimensional arrays with short-range crystalline order. Cationic membranes also support extremely efficient activation of PDE by the GTPgammaS (guanosine 5'-O-(thiotriphosphate)) form of Galpha(t) (Galpha(t)-GTPgammaS), minimizing functional heterogeneity of transducin and allowing activation with nanomolar Galpha(t)-GTPgammaS. Quantification of PDE activation and of the amount of Galpha(t)-GTPgammaS bound to PDE indicated that G(t) activates PDE maximally when bound in a 1:1 molar ratio. No cooperativity was observed, even at nanomolar concentrations. Thus, under these conditions, the one binding site for Galpha(t)-GTPgammaS on PDE that stimulates catalysis must be of higher affinity than one or more additional sites which are silent with respect to activation of PDE.     

The role of electrostatics in proton-conducting membrane protein complexes

Electrostatic interactions play a key role in the coupling of electron and proton transfer in membrane protein complexes during the conversion of the energy stored in sunlight or reduced substrates into biochemical energy via a transmembrane electrochemical proton potential. Principles of charge stabilization within membrane proteins are reviewed and discussed for photosynthetic reaction centers, cytochrome c oxidases, and diheme-containing quinol:fumarate reductases. The impact of X-ray structure-based electrostatic calculations on the functional interpretation of these structural coordinates, on providing new explanations for experimental observations, and for the design of more focused additional experiments is illustrated by a number of key examples.     

Electrostatic interactions at charged lipid membranes. Hydrogen bonds in lipid membrane surfaces

Hydrogen-bonded structures within lipid membrane surfaces are not disrupted by water and are of thermodynamic and therefore potential structural importance in biological systems.     

Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.     

Ion flux across lipid bilayer membranes with charged surfaces

Summary In this paper we derive expressions for the ion flux across lipid bilayer membranes with charged surfaces treating the membrane as a continuous phase interposed between two electrolyte solutions and calculating the ion flux with the Nernst-Planck equations. The theoretical results are compared with experiments of Seufert and Hashimoto on lipid bilayer membranes with charged surface active agents added to the membranes. If the charge of both membrane surfaces has the same sign the flux of the gegenions is greatly increased whereas the flux of the coions decreases to a small amount. For oppositely charged membrane surfaces the membrane behaves like a np semiconductor and typical rectification voltage-current characteristics are obtained.     

Improving macromolecular electrostatics calculations.

Electrostatic interactions play a key role in many aspects of protein engineering. Consequently, much effort has been put into the design of software for calculating electrostatic fields around macromolecules. We show that optimization of hydrogen bonding networks can improve both the results of pK(a) calculations and the results of electrostatic calculations performed by commonly used programs such as DelPhi. Further optimization can often be achieved by flipping the side chains of asparagine, histidine and glutamine around their chi2, chi2 and chi3 torsion angles, respectively, when this improves the local hydrogen bonding network. These optimizations are applied to some well characterized proteins: BPTI, hen egg white lysozyme and superoxide dismutase. A search for flipped residues in the PDB revealed that significant improvements in electrostatic calculations in or near the active site of enzymes can be expected for about one quarter of all enzymes in the PDB.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: Effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a β-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Insertion selectivity of antimicrobial peptide protegrin-1 into lipid monolayers: effect of head group electrostatics and tail group packing

The ability to selectively target the harmful microbial membrane over that of the host cell is one of the most important characteristics of the antimicrobial peptides (AMPs). This selectivity strongly depends on the chemical and structural properties of the lipids that make up the cell membrane. A systematic study of the initial membrane selectivity of protegrin-1 (PG-1), a beta-sheet AMP, was performed using Langmuir monolayers. Constant pressure insertion assay was used to quantify the amount of PG-1 insertion and fluorescence microscopy was employed to observe the effect of PG-1 on lipid ordering. Charge and packing properties of the monolayer were altered by using lipids with different head groups, substituting saturated with unsaturated lipid tail group(s) and incorporating spacer molecules. PG-1 inserted most readily into anionic films composed of phosphatidylglycerol (PG) and lipid A, consistent with its high selectivity for microbial membranes. It also discriminated between zwitteranionic phospholipids, inserting more readily into phosphatidylcholine (PC) monolayers than those composed of phosphatidylethanolamine, potentially explaining why PG-1 is hemolytic for PC-rich human erythrocytes and not for the PE-rich erythrocytes of ruminants. Increased packing density of the monolayer by increased surface pressure, increased tail group saturation or incorporation of dihydrocholesterol diminishes the insertion of PG-1. Fluorescence microscopy shows that lipid packing is disordered upon PG-1 insertion. However, the presence of PG-1 can still affect lipid morphology even with no observed PG-1 insertion. These results show the important role that lipid composition of the cell membrane plays in the activity of AMPs.     

Spontaneous transfer of monoacyl amphiphiles between lipid and protein surfaces.

The kinetics of transfer of natural and fluorescent nonesterified fatty acids (NEFA) and lysolecithins (lysoPC) from phospholipid and protein surfaces were measured. The kinetics of transfer of 12-(1-pyrenyl)dodecanoic acid, from liquid crystalline and gel phase single unilamellar phospholipid vesicles, very low, low, and high density lipoproteins, human serum albumin, and rat liver fatty acid-binding protein, were first-order and characterized by similar rate constants. The halftimes (t1/2) of NEFA transfer from lipids and proteins were dependent on the acyl chain structure according to log t1/2 = -0.62n + 0.59m + 12.0, where n and m, respectively, are the numbers of carbon atoms and double bonds. The structure of the donor surface had a measurable but smaller effect on transfer rates. The kinetics of NEFA and lysoPC transfer are slow relative to the lipolytic processes that liberate them. Therefore, one would predict a transient accumulation of NEFA and lysoPC during lipolysis and an attendant modulation of many metabolic processes within living cells and within the plasma compartment of blood. These data will be useful in the refinement of current models of membrane and lipoprotein function and in the selection of fluorescent NEFA analogs for studying transport in living cells.     

Beyond electrostatics: Antimicrobial peptide selectivity and the influence of cholesterol-mediated fluidity and lipid chain length on protegrin-1 activity

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1’s incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.