Time-resolved fluorescence of tryptophan characterizes membrane perturbation by cyclic lipopeptides.

Viscosin is a membrane-permeabilizing, cyclic lipopeptide (CLiP) produced by Pseudomonas species. Here, we have studied four synthetic analogs (L1W, V4W, L5W, and L7W), each with one leucine (Leu; L) or valine residue exchanged for tryptophan (Trp; W) by means of time-resolved fluorescence spectroscopy of Trp. To this end, we recorded the average fluorescence lifetime, rotational correlation time and limiting anisotropy, dipolar relaxation time and limiting extent of relaxation, rate constant of acrylamide quenching, effect of H2O-D2O exchange, and time-resolved half-width of the spectrum in the absence and presence of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) liposomes. Structure, localization, and hydration of the peptides were described by molecular dynamics simulations. The combination of the parameters provides a good description of the molecular environments of the Trp positions and the behavior of viscosin as a whole. Of particular value for characterizing the impact of viscosin on the membrane is the dipolar relaxation of Trp4 in V4W, which is deeply embedded in the hydrophobic core. The limiting relaxation level represents the membrane perturbation-unlike typical membrane probes-at the site of the perturbant. Fractions of Trp4 relax at different rates; the one not in contact with water upon excitation relaxes via recruitment of a water molecule on the 10-ns timescale. This rate is sensitive to the concerted membrane perturbation by more than one lipopeptide, which appears at high lipopeptide concentration and is assumed a prerequisite for the final formation of a membrane-permeabilizing defect. Trp7 relaxes primarily with respect to neighboring Ser residues. Trp5 flips between a membrane-inserted and surface-exposed orientation.

Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.

Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.

Eutectic Resolves Lysolipid Paradox in Thermoresponsive Liposomes.

A better molecular understanding of the temperature-triggered drug release from lysolipid-based thermosensitive liposomes (LTSLs) is needed to overcome the recent setbacks in developing this important drug delivery system. Enhanced drug release was previously rationalized in terms of detergent-like effects of the lysolipid monostearyl lysophosphatidylcholine (MSPC), stabilizing local membrane defects upon LTSL lipid melting. This is highly surprising and here referred to as the 'lysolipid paradox,' because detergents usually induce the opposite effect─they cause leakage upon freezing, not melting. Here, we aim at better answers to (i) why lysolipid does not compromise drug retention upon storage of LTSLs in the gel phase, (ii) how lysolipids can enhance drug release from LTSLs upon lipid melting, and (iii) why LTSLs typically anneal after some time so that not all drug gets released. To this end, we studied the phase transitions of mixtures of dipalmitoylphosphatidylcholine (DPPC) and MSPC by a combination of differential scanning and pressure perturbation calorimetry and identified the phase structures with small- and wide-angle X-ray scattering (SAXS and WAXS). The key result is that LTSLs, which contain the standard amount of 10 mol % MSPC, are at a eutectic point when they release their cargo upon melting at about 41 °C. The eutectic present below 41 °C consists of a MSPC-depleted gel phase as well as small domains of a hydrocarbon chain interdigitated gel phase containing some 30 mol % MSPC. In these interdigitated domains, the lysolipid is stored safely without compromising membrane integrity. At the eutectic temperature, both the MSPC-depleted bilayer and interdigitated MSPC-rich domains melt at once to fluid bilayers, respectively. Intact, fluid membranes tolerate much less MSPC than interdigitated domains─where the latter have melted, the high local MSPC content causes transient pores. These pores allow for fast drug release. However, these pores disappear, and the membrane seals again as the MSPC distributes more evenly over the membrane so that its local concentration decreases below the pore-stabilizing threshold. We provide a pseudobinary phase diagram of the DPPC-MSPC system and structural and volumetric data for the interdigitated phase.

Complex electrostatic effects on the selectivity of membrane-permeabilizing cyclic lipopeptides.

Cyclic lipopeptides (CLiPs) have many biological functions, including the selective permeabilization of target membranes, and technical and medical applications. We studied the anionic CLiP viscosin from Pseudomonas along with a neutral analog, pseudodesmin A, and the cationic viscosin-E2K to better understand electrostatic effects on target selectivity. Calcein leakage from liposomes of anionic phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) is measured in comparison with net-neutral phosphatidylcholine by time-resolved fluorescence. By contrast to the typical selectivity of cationic peptides against anionic membranes, we find viscosin more active against PG/PE at 30 µM lipid than viscosin-E2K. At very low lipid concentration, the selectivity is reversed. An equi-activity analysis reveals the reciprocal partition coefficients, 1/K, and the CLiP-to-lipid mole ratio within the membrane as leakage after 1 h reaches 50%, Re50. As expected, 1/K to PG/PE is much lower (higher affinity) for viscosin-E2K (3 µM) than viscosin (15 µM). However, the local damage to the PG/PE membrane caused by a viscosin molecule is much stronger than that of viscosin-E2K. This can be explained by the strong membrane expansion due to PG/viscosin repulsion inducing asymmetry stress between the two leaflets and, ultimately, transient limited leakage at Re50 = 0.08. PG/viscosin-E2K attraction opposes expansion and leakage starts only as the PG charges in the outer leaflet are essentially compensated by the cationic peptide (Re50 = 0.32). In the high-lipid regime (at lipid concentrations cL ≫ 1/K), virtually all CLiP is membrane bound anyway and Re50 governs selectivity, favoring viscosin. In the low-lipid regime at cL ≪ 1/K, virtually all CLiP is in solution, 1/K becomes important and the "cation attacks anionic membrane" selectivity gets restored. Overall, activity and selectivity data can only properly be interpreted if the lipid regime is known and predictions for other lipid concentrations or cell counts require knowledge of 1/K and Re50.

Vesicle budding caused by lysolipid-induced asymmetry stress.

Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20-30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a "budding power" of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20-30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively.

Extending the Pseudo-Phase Model of Detergent-Lipid Dispersions by a Detergent-Binding Protein.

Mixed micellar drug delivery systems for poorly soluble active pharmaceutical ingredients (APIs) are easy to produce and long-term stable, because they represent equilibrium structures. However, their fate after intravenous injection is still largely unknown. Once injected into the bloodstream, they can potentially convert to vesicles or disappear altogether, with both API and excipients being picked up by blood components. Our study aimed at reducing the gap between the good, quantitative understanding of aqueous glycocholate (GC)-lecithin dispersions alone and the highly complex situation in the blood. To this end, we extended the pseudophase model previously established for lipid-detergent dispersions to include the detergent-binding protein albumin as another component. The model predicted a quaternary phase diagram with planar phase boundaries defined by key parameters of the ternary subsystems, which were then determined by isothermal titration calorimetry. They include the aqueous GC concentration upon bilayer-micelle coexistence, 5.2 mM, the GC-to-lipid mole ratios in coexisting bilayers (Resat = 0.2) and micelles (Resol = 0.7), as well as the capacity of the albumin to bind 0.1 GC molecules with a dissociation constant of KD = 0.1 mM and 6 GC molecules with KD = 0.7 mM. Subsequent measurements in the quaternary system showed phase boundaries in good agreement with the model predictions. In addition, the critical micelle concentration of GC shows a minimal value (midpoint of transition) of 9.1 mM at the temperature of 24 °C where the demicellization enthalpy is zero. The demicellization process is accompanied by a heat capacity change of 29 cal/mol K. The model improves the understanding of the mixed micellar drug delivery systems. The success of the approach encourages including even more blood components, like lipoproteins, to a quantitative treatment.

Calcium affects CHP1 and CHP2 conformation and their interaction with sodium/proton exchanger 1.

Calcineurin B homologous proteins (CHPs) belong to the EF-hand Ca2+ -binding protein (EFCaBP) family. They have multiple important functions including the regulation of the Na+ /H+ exchanger 1 (NHE1). The human isoforms CHP1 and CHP2 share high sequence similarity, but have distinct expression profiles with CHP2 levels for instance increased in malignant cells. These CHPs bind Ca2+ with high affinity. Biochemical data indicated that Ca2+ can regulate their functions. Experimental evidence for Ca2+ -modulated structural changes was lacking. With a newly established fluorescent probe hydrophobicity (FPH) assay, we detected Ca2+ -induced conformational changes in both CHPs. These changes are in line with an opening of their hydrophobic pocket that binds the CHP-binding region (CBD) of NHE1. Whereas the pocket is closed in the absence of Ca2+ in CHP2, it is still accessible for the dye in CHP1. Both CHPs interacted with CBD in the presence and absence of Ca2+ . Isothermal titration calorimetry (ITC) analysis revealed high binding affinity for both CHPs to CBD with equilibrium dissociation constants (KD s) in the nanomolar range. The KD for CHP1:CBD was not affected by Ca2+ , whereas Ca2+ -depletion increased the KD 7-fold for CHP2:CBD showing a decreased affinity. The data indicate an isoform specific regulatory interaction of CHP1 and CHP2 with NHE1.

Complex Micellization Behavior of the Polysorbates Tween 20 and Tween 80.

Polysorbates (PSs, Tweens) are widely used surfactant products consisting of a sorbitan ring connecting up to four ethylene oxide (EO) chains of variable lengths, one or more of which are esterified with fatty acids of variable lengths and saturation degrees. Pharmaceutical applications include the stabilization of biologicals in solutions and the solubilization of poorly water soluble, active ingredients. This study characterizes the complex association behavior of compendial PSs PS20 and PS80, which is fundamentally different from that of single-component surfactants. To this end, a series of demicellization experiments of isothermal titration calorimetry with different PS concentrations are evaluated. Their experiment-dependent heats of titration are converted into a common function of the state of a sample, the micellar enthalpy Qm(c). These functions demonstrate that initial micelles are already present at the lowest concentrations investigated, 2 µM for PS20 and 10 µM for PS80. Initial micelles consist primarily of the surfactant species with the lowest individual critical micelle concentration (cmc). With increasing concentration, the other PS species gradually enter these micelles in the sequence of increasing individual cmc's and hydrophilic-lipophilic balance. Concentration ranges with pronounced slopes of Qm(c) can be tentatively assigned to the uptake of the major components of the PS products. Micellization and the variation of the micelle properties progress up to at least 10 mM PS. That means the published cmc values or ranges of PS20 and PS80 may be related to certain, major components being incorporated into and forming specific micelles but must not be interpreted in terms of an absence of micelles below and constant properties, e.g., the surface activity, of the micelles above these ranges. The micellization enthalpy curves differ quite substantially between PS20 and PS80 and, in a subtler fashion, between individual quality grades such as high purity, pure lauric acid/pure oleic acid, super-refined, and China grade.

Primary and Secondary Binding of Exenatide to Liposomes.

The interactions of exenatide, a Trp-containing peptide used as a drug to treat diabetes, with liposomes were studied by isothermal titration calorimetry (ITC), tryptophan (Trp) fluorescence, and microscale thermophoresis measurements. The results are not only important for better understanding the release of this specific drug from vesicular phospholipid gel formulations but describe a general scenario as described before for various systems. This study introduces a model to fit these data on the basis of primary and secondary peptide-lipid interactions. Finally, resolving apparent inconsistencies between different methods aids the design and critical interpretation of binding experiments in general. Our results show that the net cationic exenatide adsorbs electrostatically to liposomes containing anionic diacyl phosphatidylglycerol lipids (PG); however, the ITC data could not properly be fitted by any established model. The combination of electrostatic adsorption of exenatide to the membrane surface and its self-association (Kd = 46 µM) suggested the possibility of secondary binding of peptide to the first, primarily (i.e., lipid-) bound peptide layer. A global fit of the ITC data validated this model and suggested one peptide to bind primarily per five PG molecules with a Kd ≈ 0.2 µM for PC/PG 1:1 and 0.6 µM for PC/PG 7:3 liposomes. Secondary binding shows a weaker affinity and a less exothermic or even endothermic enthalpy change. Depending on the concentration of liposomes, secondary binding may also lead to liposomal aggregation as detected by dynamic light-scattering measurements. ITC quantifies primary and secondary binding separately, whereas microscale thermophoresis and Trp fluorescence represent a summary or average of both effects, possibly with the fluorescence data showing somewhat greater weighting of primary binding. Systems with secondary peptide-peptide association within the membrane are mathematically analogous to the adsorption discussed here.

Lipid Scrambling Induced by Membrane-Active Substances.

The functional roles of the lipid asymmetry of biomembranes are attracting increasing attention. This study characterizes the activity of surfactants to induce transmembrane flip-flop of lipids and thus "scramble" this asymmetry. Detergent-induced lipid scrambling of liposomes mimicking the charge asymmetry of bacterial membranes with 20 mol % of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol in the outer leaflet only was quantified by ζ-potential measurements for octaethylene glycol dodecyl ether (C12EO8), octyl glucoside (OG), and dodecyl maltoside. Membrane leakage was separately measured by the fluorescence lifetime-based calcein leakage assay and the onset of the membrane-to-micelle transition by isothermal titration calorimetry. Partition coefficients and partial molar areas were obtained as well. For the quickly membrane-permeant C12EO8 and OG, leakage proceeds at a rather sharp threshold content in the membrane, which is well below the onset of solubilization and little dependent on incubation time; it is accompanied by fast lipid scrambling. However, unlike leakage, flip-flop is a relaxation process that speeds up gradually from taking weeks in the detergent-free membrane to minutes or less in the leaking membrane. Hence, after 24 h of incubation, 10 mol % of C12EO8 or 50 mol % of OG in the membrane suffice for virtually complete lipid scrambling, whereas leakage remains below 10% for up to 14 mol % of C12EO8 and 88 mol % of OG. There is thus a concentration window in which lipid scrambling proceeds without leakage. This implies that lipid scrambling must be considered a possible mode of action of antimicrobial peptides and other membrane-active drugs or biomolecules. A related, detergent-based protocol for scrambling the lipid asymmetry of liposomes and maybe cells without compromising their overall integrity would be a very valuable tool to study functions of lipid asymmetry.

Stairway to Asymmetry: Five Steps to Lipid-Asymmetric Proteoliposomes.

Membrane proteins are embedded in a complex lipid environment that influences their structure and function. One key feature of nearly all biological membranes is a distinct lipid asymmetry. However, the influence of membrane asymmetry on proteins is poorly understood, and novel asymmetric proteoliposome systems are beneficial. To our knowledge, we present the first study on a multispanning protein incorporated in large unilamellar liposomes showing a stable lipid asymmetry. These asymmetric proteoliposomes contain the Na+/H+ antiporter NhaA from Salmonella Typhimurium. Asymmetry was introduced by partial, outside-only exchange of anionic phosphatidylglycerol (PG), mimicking this key asymmetry of bacterial membranes. Outer-leaflet and total fractions of PG were determined via ζ-potential (ζ) measurements after lipid exchange and after scrambling of asymmetry. ζ-Values were in good agreement with exclusive outside localization of PG. The electrogenic Na+/H+ antiporter was active in asymmetric liposomes, and it can be concluded that reconstitution and generation of asymmetry were successful. Lipid asymmetry was stable for more than 7 days at 23°C and thus enabled characterization of the Na+/H+ antiporter in an asymmetric lipid environment. We present and validate a simple five-step protocol that addresses key steps to be taken and pitfalls to be avoided for the preparation of asymmetric proteoliposomes: 1) optimization of desired lipid composition, 2) detergent-mediated protein reconstitution with subsequent detergent removal, 3) generation of lipid asymmetry by partial exchange of outer-leaflet lipid, 4) verification of lipid asymmetry and stability, and 5) determination of protein activity in the asymmetric lipid environment. This work offers guidance in designing asymmetric proteoliposomes that will enable researchers to compare functional and structural properties of membrane proteins in symmetric and asymmetric lipid environments.

Preparation of Asymmetric Liposomes Using a Phosphatidylserine Decarboxylase.

Lipid asymmetries between the outer and inner leaflet of the lipid bilayer exist in nearly all biological membranes. Although living cells spend great effort to adjust and maintain these asymmetries, little is known about the biophysical phenomena within asymmetric membranes and their role in cellular function. One reason for this lack of insight into such a fundamental membrane property is the fact that the majority of model-membrane studies have been performed on symmetric membranes. Our aim is to overcome this problem by employing a targeted, enzymatic reaction to prepare asymmetric liposomes with phosphatidylserine (PS) primarily in the inner leaflet. To achieve this goal, we use a recombinant version of a water soluble PS decarboxylase from Plasmodium knowlesi, which selectively decarboxylates PS in the outer leaflet, converting it to phosphatidylethanolamine. The extent of decarboxylation is quantified using high-performance thin-layer chromatography, and the local concentration of anionic PS in the outer leaflet is monitored in terms of the ζ potential. Starting, for example, with 21 mol % 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine sodium salt, the assay leads to liposomes with 21 mol % in the inner and 6 mol % PS in the outer leaflet. This asymmetry persists virtually unchanged for at least 4 days at 20°C and at least 2 days at 40°C. The use of a highly specific enzyme carries the advantage that a minor component such as PS can be adjusted without affecting or being affected by the other lipid species present in the model membrane. The phenomena governing the residual outside PS content are addressed but warrant further study.

Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition.

The asymmetric distribution of lipids between the two bilayer leaflets represents a typical feature of biological membranes. The loss of this asymmetry, for example the exposure of negatively charged lipids on the extracellular membrane leaflet of mammalian cells, is involved in apoptosis and occurs in tumor cells. Thus, the controlled production of asymmetric liposomes helps to better understand such crucial cellular processes. Here, we present an approach that allows us to design asymmetric model-membrane experiments on a rational basis and predict the fraction of exchanged lipid. In addition, we developed a label-free and nondestructive assay to quantify the asymmetric uptake of negatively charged lipids in terms of the zeta potential. This significantly enhances the applicability, impact, and predictive power of model membranes.

"Staying Out" Rather than "Cracking In": Asymmetric Membrane Insertion of 12:0 Lysophosphocholine.

Interactions between detergents and model membranes are well described by the three-stage model: saturation and solubilization boundaries divide bilayer-only, bilayer-micelle coexistence, and micelle-only ranges. An underlying assumption of the model is the equilibration of detergent between the two membrane leaflets. However, many detergents partition asymmetrically at room temperature due to slow flip-flop, such as sodium dodecyl sulfate (SDS) and lysolipids. In this work, we use isothermal titration calorimetry (ITC) and dynamic light scattering (DLS) to investigate the solubilization of unilamellar POPC vesicles by 12:0 lysophosphocholine (12:0 LPC). Flip-flop of 12:0 LPC occurs beyond the time scale of our experiments, which establish a characteristic nonequilibrated state with asymmetric distribution: 12:0 LPC partitions primarily into the outer leaflet. Increasing asymmetry stress in the membrane does not lead to membrane failure, i.e., "cracking in" as seen for alkyl maltosides and other surfactants; instead, it reduces further membrane insertion which leads to the "staying out" of 12:0 LPC in solution. At above the critical micellar concentration of 12:0 LPC in the presence of the membrane, micelles persist and accommodate further LPC but take up lipid from vesicles only very slowly. Ultimately, solubilization proceeds via the micellar mechanism (Kragh-Hansen et al., 1995). With a combination of demicellization and solubilization experiments, we quantify the molar ratio partition coefficient (0.6 ± 0.1 mM-1) and enthalpy of partitioning (6.1 ± 0.3 kJ·mol-1) and estimate the maximum detergent/lipid ratio reached in the outer leaflet (<0.13). Despite the inapplicability of the three-stage model to 12:0 LPC at room temperature, we are able to extract quantitative information from ITC solubilization experiments and DLS that are important for the understanding of asymmetry-dependent processes such as endocytosis and the gating of mechanosensitive channels in vitro.

Vesicle Leakage Reflects the Target Selectivity of Antimicrobial Lipopeptides from Bacillus subtilis.

Cyclic lipopeptides act against a variety of plant pathogens and are thus highly efficient crop-protection agents. Some pesticides contain Bacillus subtilis strains that produce lipopeptide families, such as surfactins (SF), iturins (IT), and fengycins (FE). The antimicrobial activity of these peptides is mainly mediated by permeabilizing cellular membranes. We used a fluorescence-lifetime based leakage assay to examine the effect of individual lipid components in model membranes on lipopeptide activity. Leakage induction by FE was strongly inhibited by cholesterol (CHOL) as well as by phosphatidylethanolamine (PE) and -glycerol (PG) lipids. Already moderate amounts of CHOL increased the tolerable FE content in membranes by an order of magnitude to 0.5 FE per PC + CHOL. This indicates reduced FE-lipid demixing and aggregation, which is known to be required for membrane permeabilization and explains the strong inhibition by CHOL. Ergosterol (ERG) had a weak antagonistic effect. This confirms results of microbiological tests and agrees with the fungicidal activity and selectivity of FE. SF is known to be much less selective in its antimicrobial action. In line with this, liposome leakage by SF was little affected by sterols and PE. Interestingly, PG increased SF activity and changed its leakage mechanism toward all-or-none, suggesting more specific, larger, and/or longer-lived defect structures. This may be because of the reduced energetic cost of locally accumulating anionic SF in an anionic lipid matrix. IT was found largely inactive in our assays. B. subtilis QST713 produces the lipopeptides in a ratio of 6 mol SF: 37 mol FE: 57 mol IT. Leakage induced by this native mixture was inhibited by CHOL and PE, but unaffected by ERG and by PG in the absence of PE. Note that fungi contain anionic lipids, but little PE. Hence, our data explain the strong, fungicidal activity and selectivity of B. subtilis QST713 lipopeptides.

Utilizing zeta potential measurements to study the effective charge, membrane partitioning, and membrane permeation of the lipopeptide surfactin.

The effective charge of membrane-active molecules such as the fungicidal lipopeptide surfactin (SF) is a crucial property governing solubility, membrane partitioning, and membrane permeability. We present zeta potential measurements of liposomes to measure the effective charge as well as membrane partitioning of SF by utilizing what we call an equi-activity analysis of several series of samples with different lipid concentrations. We observe an effective charge of -1.0 for SF at pH8.5 and insignificantly lower at pH7.4, illustrating that the effective charge may deviate strongly from the nominal value (-2 for 1 Asp, 1 Glu). The apparent partition coefficient decreases from roughly 100 to 20/mM with increasing membrane content of SF in agreement with the literature. Finally, by comparing zeta potentials measured soon after the addition of peptide to liposomes with those measured after a heat treatment to induce transmembrane equilibration of SF, we quantified the asymmetry of partitioning between the outer and inner leaflets. At very low concentration, SF binds exclusively to the outer leaflet. The onset of partial translocation to the inner leaflet occurs at about 5mol-% SF in the membrane. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Correlating antimicrobial activity and model membrane leakage induced by nylon-3 polymers and detergents.

Most antimicrobial peptides act upon target microorganisms by permeabilizing their membranes. The mode of action is often assessed by vesicle leakage experiments that use model membranes, with the assumption that biological activity correlates with the permeabilization of the lipid bilayer. The current work aims to extend the interpretation of vesicle leakage results and examine the correlation between vesicle leakage and antimicrobial activity. To this end, we used a lifetime-based leakage assay with calcein-loaded vesicles to study the membrane permeabilizing properties of a novel antifungal polymer poly-NM, two of its analogs, and a series of detergents. In conjunction, the biological activities of these compounds against Candida albicans were assessed and correlated with data from vesicle leakage. Poly-NM induces all-or-none leakage in polar yeast lipid vesicles at the polymer's MIC, 3 µg mL(-1). At this and higher concentrations, complete leakage after an initial lag time was observed. Concerted activity tests imply that this polymer acts independently of the detergent octyl glucoside (OG) for both vesicle leakage and activity against C. albicans spheroplasts. In addition, poly-NM was found to have negligible activity against zwitterionic vesicles and red blood cells. Our results provide a consistent, detailed picture of the mode of action of poly-NM: this polymer induces membrane leakage by electrostatic lipid clustering. In contrast, poly-MM:CO, a nylon-3 polymer comprised of both cationic and hydrophobic segments, seems to act by a different mechanism that involves membrane asymmetry stress. Vesicle leakage for this polymer is transient (limited to <100%) and graded, non-specific among zwitterionic and polar yeast lipid vesicles, additive with detergent action, and correlates poorly with biological activity. Based on these results, we conclude that comprehensive leakage experiments can provide a detailed description of the mode of action of membrane permeabilizing compounds. Without this thorough approach, it would have been logical to assume that the two nylon-3 polymers we examined act via similar mechanisms; it is surprising that their mechanisms are so distinct. Some, but not all mechanisms of vesicle permeabilization allow for antimicrobial activity.