Filling the Gap with Long n-Alkanes: Incorporation of C20 and C30 into Phospholipid Membranes.

Investigating how hydrophobic molecules mix with phospholipid bilayers and how they affect membrane properties is commonplace in biophysics. Despite this, a molecular-level empirical description of a membrane model as simple as a phospholipid bilayer with long linear hydrophobic chains incorporated is still missing. Here, we present an unprecedented molecular characterization of the incorporation of two long n-alkanes, n-eicosane (C20) and n-triacontane (C30) with 20 and 30 carbons, respectively, in phosphatidylcholine (PC) bilayers using a combination of experimental techniques (2H NMR, 31P NMR, 1H-13C dipolar recoupling solid-state NMR, X-ray scattering, and cryogenic electron microscopy) and atomistic molecular dynamics (MD) simulations. At low hydration, deuterated C20 and C30 yield 2H NMR spectra evidencing anisotropic-motion, which demonstrates their miscibility in PC membranes up to a critical alkane-to-acyl-chain volume fraction, ϕc. The acquired 2H NMR spectra of C20 and C30 have notably different lineshapes. At low alkane volume fractions below ϕc, CHARMM36 MD simulations predict such 2H NMR spectra qualitatively and thus enable an atomistic-level interpretation of the spectra. Above ϕc, the 2H NMR lineshapes become characteristic of motions in the intermediate-regime that, together with the MD simulation results, suggest the onset of immiscibility between the alkane molecules and the acyl chains. For all the systems investigated, the phospholipid molecular structure is unperturbed by the presence of the alkanes. However, at conditions of excess hydration and at surprisingly low alkane fractions below ϕc, a peak characteristic of isotropic motion is observed in both the 2H spectra of the alkanes and 31P spectra of the phospholipids, strongly indicating that the incorporation of the alkanes induces a reduction on the average radius of the lipid vesicles.

Albumin displacement at the air-water interface by Tween (Polysorbate) surfactants.

Tween (polysorbate) 20 and 80 are surfactants used for the development of parenteral protein drugs, due to their beneficial safety profile and stabilisation properties. To elucidate the mechanism by which Tween 20 and 80 stabilise proteins in aqueous solutions, either by a "direct" protein to surfactant interaction and/or by an interaction with the protein film at the air-water interface, we used spectroscopic (Infrared Reflection Absorption Spectroscopy, IRRAS) and microscopic techniques (Brewster Angle Microscopy, BAM) in combination with surface pressure measurements. To this end, the impact of both types of Tweens with regard to the displacement of the protein from the air-water interface was studied. As a model protein, human serum albumin (HSA) was used. The results for the displacement of the adsorbed HSA films by Tweens 20 and 80 can partially be understood on the basis of an orogenic displacement mechanism, which depends on the critical surface pressure of the adsorbed protein film. With increasing concentration of Tween in the sub-phase, BAM images showed the formation of different domain morphologies. IRRA-spectra supported the finding that at high protein concentration in the sub-phase, the protein film could not be completely displaced by the surfactants. Comparing the impact of both surfactants, we found that Tween 20 adsorbed faster to the protein film than Tween 80. The adsorption kinetics of both Tweens and the speed of protein displacement increased with rising surfactant concentration. Tween 80 reached significant lower surface pressures than Tween 20, which led to an incomplete displacement of the observed HSA film.

Monolayer behavior of pure F-DPPC and mixed films with DPPC studied by epifluorescence microscopy and infrared reflection absorption spectroscopy.

The monolayer behavior of a l-DPPC derivative with a single fluorination in one of its terminal methyl groups (F-DPPC) at air-water interface was investigated by epifluorescence microscopy and infrared reflection absorption spectroscopy (IRRAS). Epifluorescence microscopy was utilized to study the shape and morphology of liquid-condensed (LC) domains observed upon compression of the film. IRRAS was employed for the determination of chain order and orientation. The shapes of LC-domains in a monolayer of F-DPPC are more dependent on the rate of compression than those of DPPC. The LC domains of F-DPPC display pronounced fractal growth patterns depending on the compression speed. The evolution of LC domain occurs under dominating electrostatic dipolar forces in F-DPPC. IRRAS measurements with the analysis of the frequency of the methylene stretching vibrations as a function of film compression show that the acyl chains in an F-DPPC monolayer in the LE-phase are more disordered than those in a DPPC film. The reason for the higher chain disorder in LE phase F-DPPC monolayers is a back folding of the fluorinated sn-2 chain terminus towards the air-water interface leading to larger molecular area requirement. Angular dependent IRRA spectra of monolayers at a surface pressure of 30 mN m-1 show that in the LC phase DPPC and F-DPPC exhibit a similar tilt of the acyl chains of ca. 28-30 ° relative to the surface normal. F-DPPC is ideally miscible with l-DPPC-d62 having the same chirality, as indicated by epifluorescence images and by IRRAS. However, the LC domains in an equimolar mixture of d-DPPC and F-DPPC having opposite chirality show multi-lobed complex domain patterns indicating chiral phase separation within LC domains.

The impact of non-ideality of lipid mixing on peptide induced lipid clustering.

The influence of several antimicrobial trivalent cyclic hexapeptides on the mixing behavior of bilayer lipid membranes containing phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) with varying composition was studied using DSC and ITC. The peptides contained three arginines and three aromatic amino acids and had different sequences. All of them induce clustering of PG-rich clusters with bound peptides after binding. In a previous publication we could show that a correlation between clustering efficacy and the antimicrobial activity of the peptides exists (S. Finger et al., Biochim. Biophys. Acta 1848 (2015) 2998-3006). In the current study we investigated whether the non-ideality of the lipid mixture had any effect on the clustering efficacy and the critical peptide/lipid clustering ratio. We could show that for PG/PE membranes containing 1:1 M ratios and lipids with equal or unequal chain lengths, the amount of clustered PG depended only slightly on the absolute chain length and on the chain length difference between PG and PE. Much larger differences were observed when the PG/ PE mixing ratio was changed. In mixtures of DPPG/DPPE with high PG content, the amount of clustered PG per added peptide was much higher than in PE-rich mixtures. The ITC experiments showed that the critical peptide/lipid ratio for cluster formation is also strongly dependent on the PG/PE ratio in the mixture. In the PG/PE 3:1 mixture, the formation of clusters with bound peptide is much more likely than for mixtures with less PG. For 1:1 and 1:3 lipid mixtures, the critical peptide/lipid ratio for demixing is between 0.002 and 0.004. Therefore, even in these mixtures clustering occurs way below charge saturation of the PG in the mixture and the PG-rich clusters are not charge compensated either. The peptide concentration necessary for inducing clustering amounts to ~8 µM, a value well within the range of minimal inhibitory concentration values observed for the cyclic peptides studied here. Our results show that not only the structure of the cyclic peptide influences the clustering efficacy but also the mixing behavior of the lipids in the bilayers has an influence on the amount of clustering induced by binding of cyclic peptides.

Ligand Distribution and Lipid Phase Behavior in Phospholipid-Coated Microbubbles and Monolayers.

Phospholipid-coated targeted microbubbles are ultrasound contrast agents that can be used for molecular imaging and enhanced drug delivery. However, a better understanding is needed of their targeting capabilities and how they relate to microstructures in the microbubble coating. Here, we investigated the ligand distribution, lipid phase behavior, and their correlation in targeted microbubbles of clinically relevant sizes, coated with a ternary mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), with PEG40-stearate and DSPE-PEG2000. To investigate the effect of lipid handling prior to microbubble production in DSPC-based microbubbles, the components were either dispersed in aqueous medium (direct method) or first dissolved and mixed in an organic solvent (indirect method). To determine the lipid-phase behavior of all components, experiments were conducted on monolayers at the air/water interface. In comparison to pure DSPC and DPPC, the ternary mixtures had an additional transition plateau around 10-12 mN/m. As confirmed by infrared reflection absorption spectroscopy (IRRAS), this plateau was due to a transition in the conformation of the PEGylated components (mushroom to brush). While the condensed phase domains had a different morphology in the ternary DPPC and DSPC monolayers on the Langmuir trough, the domain morphology was similar in the coating of both ternary DPPC and DSPC microbubbles (1.5-8 µm diameter). The ternary DPPC microbubbles had a homogenous ligand distribution and significantly less liquid condensed (LC) phase area in their coating than the DSPC-based microbubbles. For ternary DSPC microbubbles, the ligand distribution and LC phase area in the coating depended on the lipid handling. The direct method resulted in a heterogeneous ligand distribution, less LC phase area than the indirect method, and the ligand colocalizing with the liquid expanded (LE) phase area. The indirect method resulted in a homogenous ligand distribution with the largest LC phase area. In conclusion, lipid handling prior to microbubble production is of importance for a ternary mixture of DSPC, PEG40-stearate, and DSPE-PEG2000.

Electrostatic interactions of alkaline earth cations with 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) model membranes at neutral and acidic pH.

The binding of alkaline earth cations Mg2+, Ca2+, and Sr2+ (M2+) to unilamellar 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid (DMPA) vesicles was analysed by pH potentiometry, differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and FT-IR spectroscopy. The binding of alkaline earth cations induces deprotonation of the DMPA headgroup even at very low concentration of divalent cations (~ 100 µM). The amount of deprotonated DMPA was measured by pH potentiometry as a function of divalent cation concentration. The thermotropic phase behaviour of DMPA:M2+ complexes was studied by DSC and FT-IR as a function of pH of the dispersion (pH 7 and pH 3-5). The formation of metastable phases was observed, especially for Ca2+ and Sr2+ at pH 3-5. In unbuffered solutions, the divalent cations bind to single and/or double negatively charged DMPA, leading to the formation of different complexes and changes in the mixing behaviour of the two complexes. At pH 7, all three equimolar lipid/cation mixtures form a very stable, highly ordered 1:1 DMPA:M2+ complex. At lower divalence, the presence of a mixture of 2:1 and 1:1 complexes was observed. FT-IR spectroscopy experiments indicated an ordering of the acyl chains of DMPA after ion binding even in the liquid-crystalline phase and the induction of the dissociation of the second proton from the headgroup induced by Ca2+ or Sr2+ binding at pH 7. With ITC, the binding enthalpy ΔH of Mg2+, Ca2+, and Sr2+ to DMPA model membranes in the gel and in the liquid-crystalline phase was measured. Evidence for dehydration of hydrophobic surfaces due to cation binding was derived from changes in heat capacity.

Nanofiber Formation and Polymerization of Bolalipids with Diacetylene-Modified Single Alkyl Chains.

The nanofiber formation in aqueous suspension of two classes of symmetric single-chain bolaamphiphiles with different polar headgroups and a diacetylene-modified alkyl chain with a length of 32, 34, and 36 C atoms was investigated by differential scanning calorimetry, transmission electron microscopy, and small-angle neutron scattering. As observed before for other bolalipids with phosphocholine (PC) and dimethyl-phosphoethanolamine (Me2PE) headgroups, the molecules form fibers when suspended in water at low temperatures but disassemble into micellar-like aggregates upon heating. The introduction of a diacetylene group in the middle of the long chain leads to a perturbation of chain packing so that this fiber-micelle transition occurs at lower temperature compared to the other bolalipids having unmodified alkyl chains. The aim of our project was the introduction of diacetylene groups into alkyl chains to be able to polymerize the fibers at low temperature. This should enhance the fiber stability and prevent the disassembly into micellar aggregates at higher temperature. Polymerization of aggregates containing diacetylene-modified bolaamphiphiles can be easily traced by UV/vis spectroscopy as colored products are formed. We found that polymerization of bolaamphiphiles with PC headgroups leads to a breakdown of most fibers into micellelike aggregates, and only some longer fibers segments are still detectable. In contrast, the use of Me2PE headgroups improves polymerizability and length of the polymerized fibers. The compound with 36 C atoms in the chain could be polymerized at low temperatures, and the fibers remained stable at least up to a temperature of 60 °C. This shows that the perturbation of the chain packing due to the diacetylene groups in the chains can be overcome by elongation of the chains, so that thermostable fibers with a diameter of the length of the bolalipid molecule can be successfully formed.

Controlling the Miscibility of X-Shaped Bolapolyphiles in Lipid Membranes by Varying the Chemical Structure and Size of the Polyphile Polar Headgroup.

Bolaamphiphiles are well-known naturally occurring structures that can increase the thermal and mechanical stability of the phospholipid membrane by incorporation in a transmembrane manner. Modifications of bolaamphiphiles to introduce particular structural elements such as a conjugated aromatic backbone and lateral side chains in the hydrophobic region lead to bolapolyphiles (BPs). We investigated the ability of BPs to form lyotropic phases in water. The BPs had an identical backbone and side chains, but different headgroup structures, leading to different abilities to act as hydrogen bond donors and acceptors. BPs with hydrophilic headgroups capable of acting as hydrogen bond donors as well as acceptors did not form lyotropic phases and were insoluble in water, independent of whether the polar groups were small or large. The extended lipophilic core structure and the multiple intermolecular hydrogen bonds between the headgroups prevented the formation of well-hydrated lyotropic aggregates. A BP with two large hydrophilic headgroups of several ethylene oxide moieties terminated by methyl groups formed sheet- and vesicle-like aggregates in water. These headgroups act only as hydrogen bond acceptors and cannot form hydrogen bonds in the absence of water. The miscibility of BPs with vesicles of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) in water and the resulting aggregate structures were also investigated. For BPs with headgroups acting as donors and acceptors of hydrogen bonds, macroscopic phase separation occurred in the mixed membranes, and two different membrane domains, a DPPC-rich one containing only little polyphile and a BP-rich one containing varying amounts of lipid, were formed. For headgroups without the ability to act as hydrogen bond donors, small BP aggregates were formed that were homogeneously distributed over the membrane. The lateral organization of BPs in lipid membranes can thus be controlled by the nature of the BP headgroup.

Interaction of Short Pentavalent Cationic Peptides with Negatively Charged DPPG Monolayers and Bilayers: Influence of Peptide Modifications on Binding.

The binding of oligopeptides with the structure (RX)4R and (KXX)4K, with X being the amino acid G or A, to lipid monolayers and bilayers of dipalmitoyl-phosphatidylglycerol (DPPG) was studied and compared to the binding effects of peptides with the structure (KX)4K. The monolayer adsorption experiments again showed the superposition of condensation effects due to charge compensation and insertion of amino acid side chains leading to expansion of the monolayer. The latter effect was enhanced when glycine was replaced by alanine. The thermotropic phase behavior of dipalmitoyl-phosphatidylglycerol (DPPG) bilayer membranes and their mixtures with short cationic model peptides was investigated by differential scanning calorimetry and infrared spectroscopy. Increasing the charge distance of the lysine residues in the series (K)5, (KG)4K, and (KGG)4K results in an upshift of the main phase transition of DPPG up to 5 K, as predicted for pure electrostatic binding. All peptides exhibit only unordered structures in bulk solution as well as when bound to DPPG bilayers. (KGG)4K additionally shows a high propensity of turn structures due to its flexibility. The exchange of glycine by alanine in (KAA)4K leads only to a marginal increase in Tm, in contrast to the binding of (KA)4K where the formation of intervesicular antiparallel ß-sheets occurs, leading to a much more pronounced stabilization of the gel phase. This shows that the sequence and flexibility of the oligopeptides has an important influence on the formation of secondary structures bound to the bilayers. Binding of (RX)4R peptides to DPPG bilayers has almost no influence on the lipid phase transition in bilayers. Here, condensation and insertion effects almost compensate, as the results of monolayer experiments show. This is due to the higher propensity of arginine side chains to insert into the lipid headgroup region.

Impact of Headgroup Asymmetry and Protonation State on the Aggregation Behavior of a New Type of Glycerol Diether Bolalipid.

In the present work, we describe the synthesis and the temperature-dependent aggregation behavior of a new class of asymmetrical glycerol diether bolalipids. These bolalipids are composed of a membrane-spanning alkyl chain with 32 carbon atoms (C32) in the sn-3 position, a methyl-branched C16 alkyl chain in the sn-2 position, and a zwitterionic phosphocholine headgroup in the sn-1 position of a glycerol moiety. The long C32 alkyl chain is terminated either by a second phosphocholine (PC-Gly(2C16Me)C32-PC) or by a phosphodimethylethanolamine headgroup (PC-Gly(2C16Me)C32-Me2PE). The temperature- and pH-dependent aggregation behavior of both lipids was studied using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS) experiments. The morphology of the formed aggregates in an aqueous suspension was visualized by transmission electron microscopy (TEM). We show that PC-Gly(2C16Me)C32-PC and PC-Gly(2C16Me)C32-Me2PE at pH 5 self-assemble into large lamellar aggregates and large lipid vesicles. Within these structures, the bolalipid molecules are probably assembled in a monolayer with fully interdigitated chains. The lipid molecules seem to be tilted with respect to the layer normal to ensure a dense packing of the alkyl chains. A temperature increase leads to a transition from a lamellar gel phase to the liquid-crystalline phase at about 28-30 °C for both bolalipids. The lamellar aggregates of PC-Gly(2C16Me)C32-Me2PE started to transform into nanofibers when the pH value of the suspension was increased to above 11. At pH 12, these nanofibers were the dominant aggregates.

Cospreading of Anionic Phospholipids with Peptides of the Structure (KX)4K at the Air-Water Interface: Influence of Lipid Headgroup Structure and Hydrophobicity of the Peptide on Monolayer Behavior.

Mixtures of anionic phospholipids (PG, PA, PS, and CL) with cationic peptides were cospread from a common organic solvent at the air-water interface. The compression of the mixed film was combined with epifluorescence microscopy or infrared reflection adsorption spectroscopy (IRRAS) to gain information on the interactions of the peptide with the different lipids. To evaluate the influence of the amino acid X of peptides with the sequence (KX)4K on the binding, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was mixed with different peptides with increasing hydrophobicity of the uncharged amino acid X. The monolayer isotherms of DPPG/(KX)4K mixtures show an increased area for the lift-off due to incorporation of the peptide into the liquid-expanded (LE) state of the lipid. The surface pressure for the transition from LE to the liquid-condensed (LC) state is slightly increased for peptides with amino acids X with moderate hydrophobicity. For the most hydrophobic peptide (KL)4K two plateaus are seen at a charge ratio PG to K of 5:1, and a strongly increased transition pressure is observed for a charge ratio of 1:1. Epifluorescence microscopy images and infrared spectroscopy show that the lower plateau corresponds to the LE-LC phase transition of the lipid. The upper plateau is connected with a squeeze-out of the peptide into the subphase. To test the influence of the lipid headgroup structure on peptide binding (KL)4K was cospread with different anionic phospholipids. The shift of the isotherm to larger areas for lift-off and to higher surface pressure for the LE-LC phase transition was observed for all tested anionic lipids. Epifluorescence microscopy reveals the formation of LC domains with extended filaments indicating a decrease in line tension due to accumulation of the peptides at the LC-domain boundaries. This effect depends on the size of the headgroup of the anionic phospholipid.

Aggregation behaviour of a single-chain, phenylene-modified bolalipid and its miscibility with classical phospholipids.

In the present work, we describe the synthesis of a single-chain, phenylene-modified bolalipid with two phosphocholine headgroups, PC-C18pPhC18-PC, using a Sonogashira cross-coupling reaction as a key step. The aggregation behaviour was studied as a function of temperature using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle neutron scattering (SANS). We show that our new bolalipid self-assembles into nanofibres, which transform into flexible nanofibres at 27 °C and further to small elongated micelles at 45 °C. Furthermore, the miscibility of the bolalipid with bilayer-forming phosphatidylcholines (DMPC, DPPC, and DSPC) was investigated by means of DSC, TEM, FTIR, and small angle X-ray scattering (SAXS). We could show that the PC-C18pPhC18-PC is partially miscible with saturated phosphatidylcholines; however, closed lipid vesicles with an increased thermal stability were not found. Instead, bilayer fragments and disk-like aggregates are formed.

The cmc-value of a bolalipid with two phosphocholine headgroups and a C24 alkyl chain: Unusual binding properties of fluorescence probes to bolalipid aggregates.

Bolalipids with a long alkyl chain and two phosphocholine polar groups self-assemble in water into two different types of aggregate structures, namely helical nanofibers at low temperature and two types of micellar aggregates at higher temperature. We tried to determine the critical aggregation concentration (cac) or critical micellar concentration (cmc) of the bolalipid tetracosane-1,24-bis(phosphocholine) (PC-C24-PC) by using different fluorescent probes. The use of pyrene or pyrene derivatives as fluorophores failed, whereas the probes 1,8-ANS and particularly bis-ANS gave consistent results. The structure of the bolalipid aggregates obviously hinders partitioning or binding of pyrene derivatives into the micellar interior, whereas 1,8-ANS and bis-ANS can bind to the surface of the aggregate structures. The observed large increase in fluorescence intensity of bis-ANS indicates that binding to the hydrophobic surface of the aggregates leads to a reduction of the dye mobility. However, binding of bis-ANS is relatively weak, so that the determination of a cac/cmc-value is difficult. Simulations of the intensity curves for PC-C24-PC lead to estimates of the cac/cmc-value of 0.3-1.0×10-6M, depending on the structure of the aggregates. Single molecule fluorescence correlation spectroscopy was used to determine the mobility of bis-ANS as a function of concentration of PC-C24-PC. The dye diffusion time and the molecular brightness are lower at low bolalipid concentration, when only free dye is present, and increase at higher concentration when bis-ANS is bound to the aggregates. The experimental cac/cmc-values are higher than those estimated, using an incremental method for the change in Gibbs free energy for micellization with n-alkyl-phosphocholines with only one polar group as a comparison. Apparently, for PC-C24-PC in micellar or fibrous aggregates, more CH2 groups are exposed to water than in a conventional micelle of an n-alkyl-phosphocholine.

Liquid-liquid phase separation of a monoclonal antibody at low ionic strength: Influence of anion charge and concentration.

Liquid-liquid phase separation (LLPS) of a monoclonal antibody solution was investigated at low ionic strength in the presence of oligovalent anions, such as citrate, trimellitate, pyromellitate and mellitate. Phase separation was observed at the isoelectric point of the antibody at pH8.7 as well as in more acidic pH regions in the presence of the tested oligovalent ions. This can be attributed to charge neutralization via binding of the oligovalent anions to the positively charged antibody. The influence of the anion concentration on liquid-liquid phase separation with respect to the net charge of the antibody was examined. Similarities to the formation of a complex coacervate were shown to apply. These findings enable us to understand the usage of excipients to rationally induce or avoid liquid-liquid phase separation at low ionic strength. Furthermore we present a method to directly examine the competition of different ions for the solvation shell, called buffer equilibration.

(Cryo)Transmission Electron Microscopy of Phospholipid Model Membranes Interacting with Amphiphilic and Polyphilic Molecules.

Lipid membranes can incorporate amphiphilic or polyphilic molecules leading to specific functionalities and to adaptable properties of the lipid bilayer host. The insertion of guest molecules into membranes frequently induces changes in the shape of the lipid matrix that can be visualized by transmission electron microscopy (TEM) techniques. Here, we review the use of stained and vitrified specimens in (cryo)TEM to characterize the morphology of amphiphilic and polyphilic molecules upon insertion into phospholipid model membranes. Special emphasis is placed on the impact of novel synthetic amphiphilic and polyphilic bolalipids and polymers on membrane integrity and shape stability.

Effect of Perfluoroalkyl Endgroups on the Interactions of Tri-Block Copolymers with Monofluorinated F-DPPC Monolayers.

We studied the interaction of amphiphilic and triphilic polymers with monolayers prepared from F-DPPC (1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine), a phospholipid with a single fluorine atom at the terminus of the sn-2 chain, an analogue of dipalmitoyl-phosphatidylcholine (DPPC). The amphiphilic block copolymers contained a hydrophobic poly(propylene oxide) block flanked by hydrophilic poly(glycerol monomethacrylate) blocks (GP). F-GP was derived from GP by capping both termini with perfluoro-n-nonyl segments. We first studied the adsorption of GP and F-GP to lipid monolayers of F-DPPC. F-GP was inserted into the monolayer up to a surface pressure Π of 42.4 mN m-1, much higher than GP (32.5 mN m-1). We then studied isotherms of lipid-polymer mixtures co-spread at the air-water interface. With increasing polymer content in the mixture a continuous shift of the onset of the liquid-expanded (LE) to liquid-condensed (LC) transition towards higher molecular and higher area per lipid molecule was observed. F-GP had a larger effect than GP indicating that it needed more space. At a Π-value of 32 mN m-1, GP was excluded from the mixed monolayer, whereas F-GP stayed in F-DPPC monolayers up to 42 mN m-1. F-GP is thus more stably anchored in the monolayer up to higher surface pressures. Images of mixed monolayers were acquired using different fluorescent probes and showed the presence of perfluorinated segments of F-GP at LE-LC domain boundaries.

An Asymmetrical Glycerol Diether Bolalipid with Protonable Phosphodimethylethanolamine Headgroup: The Impact of pH on Aggregation Behavior and Miscibility with DPPC.

Investigations regarding the self-assembly of (bola)phospholipids in aqueous media are crucial to understand the complex relationship between chemical structure of lipids and the shape and size of their aggregates in water. Here, we introduce a new asymmetrical glycerol diether bolaphospholipid, the compound Me2PE-Gly(2C16)C32-OH. This bolalipid contains a long (C32) ω-hydroxy alkyl chain bond to glycerol in the sn-3 position, a C16 alkyl chain at the sn-2 position, and a protonable phosphodimethylethanolamine (Me2PE) headgroup at the sn-1 position of the glycerol. The aggregation behavior of this bolalipid was studied as a function of temperature and pH using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) spectroscopy. We show that this bolalipid aggregates into condensed lamellar sheets in acidic milieu and in large sheet-like aggregates at neutral pH-value. By contrast, at a pH-value of 10, where the Me2PE headgroup is only partially protonated, small lipid disks with diameter 50⁻100 nm were additionally found. Moreover, the miscibility of this asymmetrical bolalipid with the bilayer-forming phosphatidylcholine DPPC was investigated by means of DSC and TEM. The incorporation of bolalipids into phospholipid membranes could result in stabilized liposomes applicable for drug delivery purposes. We show that mixtures of DPPC and Me2PE-Gly(2C16)C32-OH form large lamellar aggregates at pH of 5, 7, and 10. However, closed lipid vesicles (liposomes) with an increased thermal stability were not found.

Binding of the GTPase Sar1 to a Lipid Membrane Monolayer: Insertion and Orientation Studied by Infrared Reflection⁻Absorption Spectroscopy.

Membrane-interacting proteins are polyphilic polymers that engage in dynamic protein⁻protein and protein⁻lipid interactions while undergoing changes in conformation, orientation and binding interfaces. Predicting the sites of interactions between such polypeptides and phospholipid membranes is still a challenge. One example is the small eukaryotic GTPase Sar1, which functions in phospholipid bilayer remodeling and vesicle formation as part of the multimeric coat protein complex (COPII). The membrane interaction of Sar1 is strongly dependent on its N-terminal 23 amino acids. By monolayer adsorption experiments and infrared reflection-absorption spectroscopy (IRRAS), we elucidate the role of lipids in inducing the amphipathicity of this N-terminal stretch, which inserts into the monolayer as an amphipathic helix (AH). The AH inserting angle is determined and is consistent with the philicities and spatial distribution of the amino acid monomers. Using an advanced method of IRRAS data evaluation, the orientation of Sar1 with respect to the lipid layer prior to the recruitment of further COPII proteins is determined. The result indicates that only a slight reorientation of the membrane-bound Sar1 is needed to allow coat assembly. The time-course of the IRRAS analysis corroborates a role of slow GTP hydrolysis in Sar1 desorption from the membrane.

Binding of cationic model peptides (KX)4K to anionic lipid bilayers: Lipid headgroup size influences secondary structure of bound peptides.

Differential Scanning Calorimetry (DSC) and Fourier transformed Infrared (FT-IR) spectroscopy were used to test the influence of acyl chain length, acyl chain saturation, and chemical structure of anionic phospholipids on the interaction with cationic model peptides (KX)4K, with amino acid X=A, Abu, and L. The lipids used were phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidic acid (PA), and cardiolipin (CL). DSC was used to monitor the phase transition of lipid vesicles before and after peptide binding. The electrostatic attraction is the main driving force for binding. The hydrophobicity of the amino acid X influences the binding strength as well as the secondary structure of the bound peptide. Binding of peptides leads to an upshift of the lipid phase transition. Lipids with smaller headgroups show a larger upshift of the main phase transition temperature. Data from FT-IR spectroscopy show in addition that the stability of the bound ß-sheets of (KX)4K depends on the hydrophobicity of the uncharged amino acid X and on the size of the lipid headgroup. For lipids with large anionic headgroups, such as PS, the antiparallel ß-sheet of (KAbu)4K bound to gel phase bilayers is converted to an unordered structure upon heating through the lipid phase transition. Reducing the size of the headgroup, as in PG, increases the stability of the bound peptide ß-sheets. For the smallest headgroups, present in PA and CL, stably bound ß-sheets are observed even above the lipid phase transition.

Perfluorinated Moieties Increase the Interaction of Amphiphilic Block Copolymers with Lipid Monolayers.

The interaction of amphiphilic and triphilic block copolymers with lipid monolayers has been studied. Amphiphilic triblock copolymer PGMA20-PPO34-PGMA20 (GP) is composed of a hydrophobic poly(propylene oxide) (PPO) middle block that is flanked by two hydrophilic poly(glycerol monomethacrylate) (PGMA) side blocks. The attachment of a perfluoro-n-nonyl residue (F9) to either end of GP yields a triphilic polymer with the sequence F9-PGMA20-PPO34-PGMA20-F9 (F-GP). The F9 chains are fluorophilic, i.e., they have a tendency to demix in hydrophilic as well as in lipophilic environments. We investigated (i) the adsorption of both polymers to differently composed lipid monolayers and (ii) the compression behavior of mixed polymer/lipid monolayers. The lipid monolayers are composed of phospholipids with PC or PE headgroups and acyl chains of different length and saturation. Both polymers interact with lipid monolayers by inserting their hydrophobic moieties (PPO, F9). The interaction is markedly enhanced in the presence of F9 chains, which act as membrane anchors. GP inserts into lipid monolayers up to a surface pressure of 30 mN/m, whereas F-GP inserts into monolayers at up to 45 mN/m, suggesting that F-GP also inserts into lipid bilayer membranes. The adsorption of both polymers to lipid monolayers with short acyl chains is favored. Upon compression, a two-step squeeze-out of F-GP occurs, with PPO blocks being released into the aqueous subphase at 28 mN/m and the F9 chains being squeezed out at 48 mN/m. GP is squeezed out in one step at 28 mN/m because of the lack of F9 anchor groups. The liquid expanded (LE) to liquid condensed (LC) phase transition of DPPC and DMPE is maintained in the presence of the polymers, indicating that the polymers can be accommodated in LE- and LC-phase monolayers. These results show how fluorinated moieties can be included in the rational design of membrane-binding polymers.