Membrane nanotubes transform into double-membrane sheets at condensate droplets.

Cellular membranes exhibit a multitude of highly curved morphologies such as buds, nanotubes, cisterna-like sheets defining the outlines of organelles. Here, we mimic cell compartmentation using an aqueous two-phase system of dextran and poly(ethylene glycol) encapsulated in giant vesicles. Upon osmotic deflation, the vesicle membrane forms nanotubes, which undergo surprising morphological transformations at the liquid-liquid interfaces inside the vesicles. At these interfaces, the nanotubes transform into cisterna-like double-membrane sheets (DMS) connected to the mother vesicle via short membrane necks. Using super-resolution (stimulated emission depletion) microscopy and theoretical considerations, we construct a morphology diagram predicting the tube-to-sheet transformation, which is driven by a decrease in the free energy. Nanotube knots can prohibit the tube-to-sheet transformation by blocking water influx into the tubes. Because both nanotubes and DMSs are frequently formed by cellular membranes, understanding the formation and transformation between these membrane morphologies provides insight into the origin and evolution of cellular organelles.

Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane.

Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.

Role of interfacial hydrophobicity in antimicrobial peptide magainin 2-induced nanopore formation.

Antimicrobial peptide magainin 2 (Mag) forms nanopores in lipid bilayers and induces membrane permeation of the internal contents from vesicles. The binding of Mag to the membrane interface of a giant unilamellar vesicle (GUV) increases its fractional area change, δ, which is one of the main causes of Mag-induced nanopore formation. However, the role of its amino acid composition in the Mag-induced area increase and the following nanopore formation is not well understood. Here, to elucidate it we examined the role of interfacial hydrophobicity of Mag in its nanopore formation activity by investigating de novo-designed Mag mutants-induced nanopore formation in GUVs. Aligned amino acid residues in the α-helix of Mag were replaced to create 3 mutants: F5A-Mag, A9F-Mag, and F5,12,16A-Mag. These mutants have different interfacial hydrophobicity due to the variation of the numbers of Phe and Ala because the interfacial hydrophobicity of Phe is higher than that of Ala. The rate constant of Mag mutant-induced nanopore formation, kp, increased with increasing numbers of Phe residues at the same peptide concentration. Further, the Mag mutant-induced δ increased with increasing numbers of Phe residues at the same peptide concentration. These results indicate that kp and δ increase with increasing interfacial hydrophobicity of Mag mutants. The relationship between kp and δ in the Mag and its mutants clearly indicates that kp increases with increasing δ, irrespective of the difference in mutants. Based on these results, we can conclude that the interfacial hydrophobicity of Mag plays an important role in its nanopore formation activity.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

Light-Triggered Cargo Loading and Division of DNA-Containing Giant Unilamellar Lipid Vesicles.

A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-containing giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidation upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-containing GUV with full spatiotemporal control-proving the relevance of the division mechanism for bottom-up synthetic biology.

Block Copolymer Giant Unilamellar Vesicles for High-Throughput Screening.

Bottom-up synthetic cells offer the potential to study cellular processes with reduced complexity. Giant unilamellar vesicles (GUVs) can mimic cells in their morphological characteristics because their architecture is precisely controllable. We propose a block copolymer-based GUV system that can be used for high-throughput screening. Through droplet microfluidic methods, we produce double emulsions that then serve as templates for GUVs with adjustable inner, polymer membrane, and outer composition. Using flow cytometry, we are able to analyze tens of thousands of GUVs in a short amount of time, enabling their use for screening assays.

Ceiling impact on air disinfection performance of Upper-Room Germicidal Ultraviolet (UR-GUV).

This study used Computational Fluid Dynamics (CFD) to investigate air disinfection for SARS-CoV-2 by the Upper-Room Germicidal Ultraviolet (UR-GUV), with focus on ceiling impact. The study includes three indoor settings, i.e., low (airport bus), medium (classroom) and high (rehearsal room) ceilings, which were ventilated with 100% clean air (CA case), 80% air-recirculation with a low filtration (LF case), and 80% air-recirculation with a high filtration (HF case). According to the results, using UR-GUV can offset the increased infection risk caused by air recirculation, with viral concentrations in near field (NF) and far field (FF) in the LF case similar to those in the CA case. In the CA case, fraction remaining (FR) was 0.48-0.73 with 25% occupancy rate (OR) and 0.49-0.91 with 45% OR in the bus, 0.41 in NF and 0.11 in FF in the classroom, and 0.18 in NF and 0.09 in FF in the rehearsal room. Obviously, UR-GUV performance in NF can be improved in a room with a high ceiling where FR has a power relationship with UV zone height. As using UR-GUV can only extend the exposure time to get infection risk of 1% (T 1% ) to 8 min in NF in the classroom, and 47 min in NF in the rehearsal room, it is necessary to abide by social distancing in the two rooms. In addition, T 1% in FF was calculated to be 18.3 min with 25% OR and 21.4% with 45% OR in the airport bus, showing the necessity to further wear a mask.

Division and Regrowth of Phase-Separated Giant Unilamellar Vesicles*.

Success in the bottom-up assembly of synthetic cells will depend on strategies for the division of protocellular compartments. Here, we describe the controlled division of phase-separated giant unilamellar lipid vesicles (GUVs). We derive an analytical model based on the vesicle geometry, which makes four quantitative predictions that we verify experimentally. We find that the osmolarity ratio required for division is 2 , independent of the GUV size, while asymmetric division happens at lower osmolarity ratios. Remarkably, we show that a suitable osmolarity change can be triggered by water evaporation, enzymatic decomposition of sucrose or light-triggered uncaging of CMNB-fluorescein. The latter provides full spatiotemporal control, such that a target GUV undergoes division whereas the surrounding GUVs remain unaffected. Finally, we grow phase-separated vesicles from single-phased vesicles by targeted fusion of the opposite lipid type with programmable DNA tags to enable subsequent division cycles.

The ESCRT protein CHMP2B acts as a diffusion barrier on reconstituted membrane necks.

Endosomal sorting complexes required for transport (ESCRT)-III family proteins catalyze membrane remodeling processes that stabilize and constrict membrane structures. It has been proposed that stable ESCRT-III complexes containing CHMP2B could establish diffusion barriers at the post-synaptic spine neck. In order to better understand this process, we developed a novel method based on fusion of giant unilamellar vesicles to reconstitute ESCRT-III proteins inside GUVs, from which membrane nanotubes are pulled. The new assay ensures that ESCRT-III proteins polymerize only when they become exposed to physiologically relevant membrane topology mimicking the complex geometry of post-synaptic spines. We establish that CHMP2B, both full-length and with a C-terminal deletion (ΔC), preferentially binds to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Moreover, we show that CHMP2B preferentially accumulates at the neck of membrane nanotubes, and provide evidence that CHMP2B-ΔC prevents the diffusion of PI(4,5)P2 lipids and membrane-bound proteins across the tube neck. This indicates that CHMP2B polymers formed at a membrane neck may function as a diffusion barrier, highlighting a potential important function of CHMP2B in maintaining synaptic spine structures.

Membrane Engineering: Phase Separation in Polymeric Giant Vesicles.

Cell membranes exhibit elaborate lipidic patterning to carry out a myriad of functions such as signaling and trafficking. Domain formation in giant unilamellar vesicles (GUVs) is thus of interest for understanding fundamental biological processes and to provide new prospects for biocompatible soft materials. Lipid rearrangements in lipidic GUVs and lipid/polymer GUVs are extensively studied whereas polymer/polymer hybrid GUVs remain evasive. Here, the focus is on the thermodynamically driven phase separation of amphiphilic polymers in GUVs. It is demonstrated that polymer phase separation is entropically dictated by hydrophobic block incompatibilities and that films topology can help to determine the outcome of polymeric phase separation in GUVs. Lastly, Janus-GUVs are obtained and GUVs exhibit a single large domain by using a compatibilizing hydrophobic block copolymer.

Study of molecular transport through a single nanopore in the membrane of a giant unilamellar vesicle using COMSOL simulation.

The antimicrobial peptide (AMP) magainin 2 induces nanopores in the lipid membranes of giant unilamellar vesicles (GUVs), as observed by the leakage of water-soluble fluorescent probes from the inside to the outside of GUVs through the pores. However, molecular transport through a single nanopore has not been investigated in detail yet and is studied in the present work by simulation. A single pore was designed in the membrane of a GUV using computer-aided design software. Molecular transport, from the outside to the inside of GUV through the nanopore, of various fluorescent probes such as calcein, Texas-Red Dextran 3000 (TRD-3k), TRD-10k and TRD-40k was then simulated. The effect of variation in GUV size (diameter) was also investigated. A single exponential growth function was fitted to the time course of the fluorescence intensity inside the GUV and the corresponding rate constant of molecular transport was calculated, which decreases with an increase in the size of fluorescent probe and also with an increase in the size of GUV. The rate constant found by simulation agrees reasonably well with reported experimental results for inside-to-outside probe leakage. Based on Fick's law of diffusion an analytical treatment is developed for the rate constant of molecular transport that supports the simulation results. These investigations contribute to a better understanding of the mechanism of pore formation using various membrane-active agents in the lipid membranes of vesicles and the biomembranes of cells.

Key Process and Factors Controlling the Direct Translocation of Cell-Penetrating Peptide through Bio-Membrane.

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh1, which has punctured bilayer morphologies.

Effect of Vesicle Size on the Cytolysis of Cell-Penetrating Peptides (CPPs).

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle's size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs.

Spontaneous and Stress-Induced Pore Formation in Membranes: Theory, Experiments and Simulations.

The large plasticity, dynamics and adaptability of biological membranes allow different modes of intrinsic and inducible permeability. These phenomena are of physiological importance for a number of natural functions related to cell death and can also be manipulated artificially for practical purposes like gene transfer, drug delivery, prevention of infections or anticancer therapy. For these advances to develop in a controllable and specific way, we need a sufficient understanding of the membrane permeability phenomena. Since the formulation of early concepts of pore formation, there has been an enormous effort to describe membrane permeability by using theory, simulations and experiments. A major breakthrough has come recently through theoretical developments that allow building continuous trajectories of pore formation both in the absence and presence of stress conditions. The new model provides a coherent quantitative view of membrane permeabilization, useful to test the impact of known lipid properties, make predictions and postulate specific pore intermediates that can be studied by simulations. For example, this theory predicts unprecedented dependencies of the line tension on the pore radius and on applied lateral tension which explain previous puzzling results. In parallel, important concepts have also come from molecular dynamics simulations, of which the role of water for membrane permeabilization is of special interest. These advances open new challenges and perspectives for future progress in the study of membrane permeability, as experiments and simulations will need to test the theoretical predictions, while theory achieves new refinements that provide a physical ground for observations.

An Integrated Microfluidic Platform for Quantifying Drug Permeation across Biomimetic Vesicle Membranes.

The low membrane permeability of candidate drug molecules is a major challenge in drug development, and insufficient permeability is one reason for the failure of antibiotic treatment against bacteria. Quantifying drug transport across specific pathways in living systems is challenging because one typically lacks knowledge of the exact lipidome and proteome of the individual cells under investigation. Here, we quantify drug permeability across biomimetic liposome membranes, with comprehensive control over membrane composition. We integrate the microfluidic octanol-assisted liposome assembly platform with an optofluidic transport assay to create a complete microfluidic total analysis system for quantifying drug permeability. Our system enables us to form liposomes with charged lipids mimicking the negative charge of bacterial membranes at physiological pH and salt concentrations, which proved difficult with previous liposome formation techniques. Furthermore, the microfluidic technique yields an order of magnitude more liposomes per experiment than previous assays. We demonstrate the feasibility of the assay by determining the permeability coefficient of norfloxacin and ciprofloxacin across biomimetic liposomes.

Light-Guided Motility of a Minimal Synthetic Cell.

Cell motility is an important but complex process; as cells move, new adhesions form at the front and adhesions disassemble at the back. To replicate this dynamic and spatiotemporally controlled asymmetry of adhesions and achieve motility in a minimal synthetic cell, we controlled the adhesion of a model giant unilamellar vesicle (GUV) to the substrate with light. For this purpose, we immobilized the proteins iLID and Micro, which interact under blue light and dissociate from each other in the dark, on a substrate and a GUV, respectively. Under blue light, the protein interaction leads to adhesion of the vesicle to the substrate, which is reversible in the dark. The high spatiotemporal control provided by light, allowed partly illuminating the GUV and generating an asymmetry in adhesions. Consequently, the GUV moves into the illuminated area, a process that can be repeated over multiple cycles. Thus, our system reproduces the dynamic spatiotemporal distribution of adhesions and establishes mimetic motility of a synthetic cell.

Mechanism of Binding of Antifungal Antibiotic Amphotericin B to Lipid Membranes: An Insight from Combined Single-Membrane Imaging, Microspectroscopy, and Molecular Dynamics.

Amphotericin B is a lifesaving polyene antibiotic used in the treatment of systemic mycoses. Unfortunately, the pharmacological applicability of this drug is limited because of its severe toxic side effects. At the same time, the lack of a well-defined mechanism of selectivity hampers the efforts to rationally design safer derivatives. As the drug primarily targets the biomembranes of both fungi and humans, new insights into the binding of amphotericin B to lipid membranes can be helpful in unveiling the molecular mechanisms underlying both its pharmacological activity and toxicity. We use fluorescence-lifetime-imaging microscopy combined with fluorescence-emission spectroscopy in the microscale to study the interaction of amphotericin B with single lipid bilayers, using model systems based on giant unilamellar liposomes formed with three lipids: dipalmitoylphosphatidylcholine (DPPC), dimirystoylphosphatidylcholine (DMPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). The results show that amphotericin B introduced into the water phase as a DMSO solution binds to the membrane as dimers and small-molecular aggregates that we identify as tetramers and trimers. Fluorescence-detected linear-dichroism measurements revealed high orientational freedom of all the molecular-organization forms with respect to the membrane plane, which suggests that the drug partially binds to the membrane surface. The presence of sterols in the lipid phase (cholesterol but particularly ergosterol at 30 mol %) promotes the penetration of drug molecules into the lipid membrane, as concluded on the basis of the decreased orientation angle of amphotericin B molecules with respect to the axis normal to the membrane plane. Moreover, ergosterol facilitates the association of amphotericin B dimers into aggregated structures that can play a role in membrane destabilization or permeabilization. The presence of cholesterol inhibits the formation of small aggregates in the lipid phase of liposomes, making this system a promising candidate for a low-toxicity antibiotic-delivery system. Our conclusions are supported with molecular simulations that reveal the conformational properties of AmB oligomers in both aqueous solution and lipid bilayers of different compositions.

Structural analysis of phosphatidylinositol 4-kinase IIIß (PI4KB) - 14-3-3 protein complex reveals internal flexibility and explains 14-3-3 mediated protection from degradation in vitro.

Phosphatidylinositol 4-kinase IIIß (PI4KB) is responsible for the synthesis of the Golgi and trans-Golgi network (TGN) pool of phosphatidylinositol 4-phospahte (PI4P). PI4P is the defining lipid hallmark of Golgi and TGN and also serves as a signaling lipid and as a precursor for higher phosphoinositides. In addition, PI4KB is hijacked by many single stranded plus RNA (+RNA) viruses to generate PI4P-rich membranes that serve as viral replication organelles. Given the importance of this enzyme in cells, it has to be regulated. 14-3-3 proteins bind PI4KB upon its phosphorylation by protein kinase D, however, the structural basis of PI4KB recognition by 14-3-3 proteins is unknown. Here, we characterized the PI4KB:14-3-3 protein complex biophysically and structurally. We discovered that the PI4KB:14-3-3 protein complex is tight and is formed with 2:2 stoichiometry. Surprisingly, the enzymatic activity of PI4KB is not directly modulated by 14-3-3 proteins. However, 14-3-3 proteins protect PI4KB from proteolytic degradation in vitro. Our structural analysis revealed that the PI4KB:14-3-3 protein complex is flexible but mostly within the disordered regions connecting the 14-3-3 binding site of the PI4KB with the rest of the PI4KB enzyme. It also predicted no direct modulation of PI4KB enzymatic activity by 14-3-3 proteins and that 14-3-3 binding will not interfere with PI4KB recruitment to the membrane by the ACBD3 protein. In addition, the structural analysis explains the observed protection from degradation; it revealed that several disordered regions of PI4KB become protected from proteolytical degradation upon 14-3-3 binding. All the structural predictions were subsequently biochemically validated.

Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study.

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3ß,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms.

We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.