Phosphocholine Membranes Research Articles

Cholesterol Depletion and Membrane Deformation by MeβCD and the Resultant Enhanced T Cell Killing.

Recent studies have demonstrated the crucial role of cholesterol (Chol) in regulating the mechanical properties and biological functions of cell membranes. Methyl-β-cyclodextrin (MeβCD) is commonly utilized to modulate the Chol content in cell membranes, but there remains a lack of a comprehensive understanding. In this study, using a range of different techniques, we find that the optimal ratio of MeβCD to Chol for complete removal of Chol from a phosphocholine (PC)/Chol mixed membrane with a 1:1 mol ratio is 4.5:1, while the critical MeβCD-to-Chol ratio for membrane permeation falls within the range between 1.5 and 2.4. MeβCD at elevated concentrations induces the formation of fibrils or tubes from a PC membrane. Single lipid tracking reveals that removing Chol restores the diffusion of lipid molecules in the PC/Chol membrane to levels observed in pure PC membranes. Exposure to 5 mM MeβCD for 30 min effectively eliminates Chol from various cell lines, leading to an up to 8-fold enhancement in melittin cytotoxicity over Hela cells and an up to 3.5-fold augmentation of T cell cytotoxicity against B16F10-OVA cells. This study presents a diagram that delineates the concentration- and time-dependent distribution of MeβCD-induced Chol depletion and membrane deformation, which holds significant potential for modulating the mechanical properties of cellular membranes in prospective biomedical applications.

Cholesterol-Mediated Clustering of the HIV Fusion Protein gp41 in Lipid Bilayers

The envelope glycoprotein (Env) of the human immunodeficient virus (HIV-1) is known to cluster on the viral membrane surface to attach to target cells and cause membrane fusion for HIV-1 infection. However, the molecular structural mechanisms that drive Env clustering remain opaque. Here, we use solid-state NMR spectroscopy and molecular dynamics (MD) simulations to investigate nanometer-scale clustering of the membrane-proximal external region (MPER) and transmembrane domain (TMD) of gp41, the fusion protein component of Env. Using 19F solid-state NMR experiments of mixed fluorinated peptides, we show that MPER-TMD trimers form clusters with interdigitated MPER helices in cholesterol-containing membranes. Inter-trimer 19F-19F cross peaks, which are indicative of spatial contacts within ∼2 nm, are observed in cholesterol-rich virus-mimetic membranes but are suppressed in cholesterol-free model membranes. Water-peptide and lipid-peptide cross peaks in 2D 1H-19F correlation spectra indicate that the MPER is well embedded in model phosphocholine membranes but is more exposed to the surface of the virus-mimetic membrane. These experimental results are reproduced in coarse-grained and atomistic molecular dynamics simulations, which suggest that the effects of cholesterol on gp41 clustering is likely via indirect modulation of the MPER orientation. Cholesterol binding to the helix-turn-helix region of the MPER-TMD causes a parallel orientation of the MPER with the membrane surface, thus allowing MPERs of neighboring trimers to interact with each other to cause clustering. These solid-state NMR data and molecular dynamics simulations suggest that MPER and cholesterol cooperatively govern the clustering of gp41 trimers during virus-cell membrane fusion.

Stiffening of Phosphocholine Membranes by Cholesterol

Cholesterol is a key molecule in controlling membrane fluidity, packing state, and various functions in eukaryotic cell membranes. It also regulates antibiotic drug resistance by augmenting the mechanical properties of its host membrane against structural damage. While, structurally, cholesterol exhibits universal condensing effects on fluid lipid membranes (saturated and unsaturated), its effects on membrane mechanics are presumed to be non-universal. Specifically, cholesterol causes strong stiffening in saturated lipid membranes, yet its mechanical effects on unsaturated lipid membranes remain controversial. Previous studies on di-monounsaturated lipids, such as DOPC, have shown that cholesterol has little effect on the bending rigidity of such membranes, despite clear evidence of a condensing effect. In contrast, neutron spin echo (NSE) spectroscopy shows that cholesterol yields a non-trivial stiffening of DOPC membranes over NSE timescales [1]. Here, we report similar observations using solid-state 2H-NMR spectroscopy. The model-free functional dependence of spin lattice relaxation rates (R1Z) on segmental order parameters (SCD), unambiguously indicates increased membrane bending rigidity with cholesterol [2]. These results are contextualized within the fluctuation-dissipation theorem which connects relaxations from thermal fluctuations in liquid-crystalline membranes to quasi-elastic deformation on mesoscopic length scales. Parallel studies on membranes with different lipid unsaturation (e.g. POPC vs. DOPC) indicate that cholesterol-induced stiffening is dependent on chain unsaturation and is present in all studied membranes. Our dynamical results - as well as structural SANS/SAXS and ESR data - are in excellent agreement with real-space fluctuation analysis from molecular dynamics simulations on cognate DOPC-cholesterol bilayers and with standard theories of membrane mechanics. Our findings have significant biological implications and encourage reassessment of mechanical measurements of cholesterol-containing membranes over length and time scales relevant to biological processes. [1] R. Ashkar et al. Biophys. J. (2019) 116, 328a. [2] T.R. Molugu et al. (2016) Chem. Phys. Lipids 1848, 246.

Preferential binding and re-organization of nanoscale domains on model lipid membranes by pore-forming toxins: insight from STED-FCS

Potential nanodomains in cellular membranes are widely believed to be targeted by proteins and other biomolecules to enable execution of critical cellular functions. However, characterization of these nanodomains remains elusive, primarily due to the diffraction limit of a conventional optical microscope. Herein using super-resolution STED microscopy coupled with fluorescence correlation spectroscopy (STED-FCS), we provide experimental evidence of lipid nanodomains present in model membranes comprised of phosphocholine-cholesterol binary mixture, and its reorganization induced by pore-forming toxins (PFTs). In this study, we used two different types of PFTs, namely α-PFT (cytolysin A) and β-PFT (listeriolysin O), that preferentially associate as well as reorganize cholesterol-containing saturated and unsaturated phosphocholine membranes. The emergence of nanodomains due to the lipid–lipid and lipid-PFT interactions was quantified at a length scales of ~50–150 nm using FCS diffusion law enabled by variation of spot sizes in the super-resolution STED microscopy. Our results shed light on the usefulness of super-resolution microscopy to quantify the underlying nanoscale domains in model membranes with implications for a wide variety of membrane-mediated cellular events observed in real cell membranes.

Hydrophobic Mismatch Modulates Stability and Plasticity of Human Mitochondrial VDAC2

The human mitochondrial outer membrane protein voltage-dependent anion channel isoform 2 (hVDAC2) is a β-barrel metabolite flux channel that is indispensable for cell survival. It is well established that physical forces imposed on a transmembrane protein by its surrounding lipid environment decide protein structure and stability. Yet, how the mitochondrial membrane and protein-lipid interplay together regulate hVDAC2 stability is unknown. Here, we combine experimental biophysical investigations of protein stability with all-atom molecular dynamics simulations to study the effect of the most abundant mitochondrial phosphocholine (PC) lipids on hVDAC2. We demonstrate experimentally that increasing the PC lipid acyl chain length from diC14:0 to diC18:0-PC has a nonlinear effect on the β-barrel. We show that protein stability is highest in diC16:0-PC, which exhibits a negative mismatch with the hVDAC2 barrel. Our simulations also reveal that structural rigidity of hVDAC2 is highest under optimal negative mismatch provided by diC16:0-PC bilayers. Further, we validate our observations by altering the physical properties of PC membranes indirectly using cholesterol. We propose that VDAC plasticity and stability in the mitochondrial outer membrane are modulated by physical properties of the bilayer.

Antimicrobial peptide dendrimer interacts with phosphocholine membranes in a fluidity dependent manner: A neutron reflection study combined with molecular dynamics simulations

The interaction mechanism of a novel amphiphilic antimicrobial peptide dendrimer, BALY, with model lipid bilayers was explored through a combination of neutron reflection and molecular dynamics simulations. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phos-phocholine (DPPC) lipid bilayers were examined at room temperature to extract information on the interaction of BALY with fluid and gel phases, respectively. Furthermore, a 1:4 mixture of POPC and DPPC was used as a model of a phase-separated membrane. Upon interaction with fluid membranes, BALY inserted in the distal leaflet and caused thinning and disordering of the headgroups. Membrane thinning and expansion of the lipid cross-sectional area were observed for gel phase membranes, also with limited insertion to the distal leaflet. However, dendrimer insertion through the entire lipid tail region was observed upon crossing the lipid phase transition temperature of DPPC and in phase separated membranes. The results show clear differences in the interaction mechanism of the dendrimer depending on the lipid membrane fluidity, and suggest a role for lipid phase separation in promoting its antimicrobial activity.

Amphiphilic Drug-Like Molecules Accumulate in a Membrane below the Head Group Region

The partitioning behavior of drug-like molecules into biomembranes has a crucial impact on the design and efficacy of therapeutic drugs. Thermodynamic properties connected with the interaction of molecules with membranes can be evaluated by calculating free-energy profiles normal to the membrane surface. We calculated the free-energy profiles of 25 drug-like molecules in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane and free energies of solvation in water and heptane using two methods, molecular dynamics (MD) simulations with the Berger lipid force field and COSMOmic, based on a continuum conductor-like screening model for realistic solvation (COSMO-RS). The biased MD simulations (in total ∼22 μs long) were relatively computationally expensive, whereas the COSMOmic approach offered a significantly less expensive alternative. Both methods provided similar results and showed that the studied amphiphilic drug-like molecules accumulate in the membrane, with the majority localized below the head group region. The MD simulations were more lipophilic and gave free-energy profiles that were systematically deeper than those calculated by COSMOmic. To investigate the physical nature of the increased lipophilicity, we analyzed a water/heptane system and identified that it is most likely caused by overestimation of the attractive term of the Lennard-Jones potential in lipid tails. We concluded that COSMOmic can be successfully used for high-throughput computations of global thermodynamic properties, for example, partition coefficients and energy barrier heights, in phosphocholine membranes. In contrast, MD is better for investigating local properties like molecular positioning and orientation in the membrane because they more accurately reflect the complex structure of lipid bilayers. MD is also useful for studies of highly complex systems, for example, drug-membrane-protein interactions.

Membrane-Dependent Conformation, Dynamics, and Lipid Interactions of the Fusion Peptide of the Paramyxovirus PIV5 from Solid-State NMR

The entry of enveloped viruses into cells requires protein-catalyzed fusion of the viral and cell membranes. The structure–function relation of a hydrophobic fusion peptide (FP) in viral fusion proteins is still poorly understood. We report magic-angle-spinning solid-state NMR results of the membrane-bound conformation, dynamics, and lipid interactions of the FP of the F protein of the paramyxovirus, parainfluenza virus 5 (PIV5). 13C chemical shifts indicate that the PIV5 FP structure depends on the composition of the phospholipid membrane: the peptide is α-helical in palmitoyloleoylphosphatidylglycerol-containing anionic membranes but mostly β-sheet in neutral phosphocholine membranes. Other environmental factors, including peptide concentration, cholesterol, membrane reconstitution protocol, and a Lys solubility tag, do not affect the secondary structure. The α-helical and β-sheet states exhibit distinct dynamics and lipid interactions. The β-sheet FP is immobilized, resides on the membrane surface, and causes significant membrane curvature. In contrast, the α-helical FP undergoes intermediate-timescale motion and maintains the lamellar order of the membrane. Two-dimensional 31P–1H correlation spectra show clear 31P–water cross peaks for anionic membranes containing the α-helical FP but weak or no 31P–water cross peak for neutral membranes containing the β-sheet FP. These results suggest that the β-sheet FP may be associated with high-curvature dehydrated fusion intermediates, while the α-helical state may be associated with the extended prehairpin state and the post-fusion state. Conformational plasticity is also a pronounced feature of the influenza and human immunodeficiency virus FPs, suggesting that these Gly-rich sequences encode structural plasticity to generate and sense different membrane morphologies.

Coexisting Phases in PEGylated Phosphocholine Membranes: A Model Study

Understanding the phase behavior of PEGylated phosphocholine membranes is becoming increasingly important in many biomedical applications. Here, we used binary mixtures of phosphocholines and PEG-phospholipids in monolayers on phosphate buffered saline as ideal models of PEGylated phosphocholine membranes. Several phase states and transitions between homogeneously mixed and completely immiscible phases have been visualized in these mixtures by epifluorescence microscopy, which is neither predicted nor easily explained by the existing interpretive schemes. The results of our study suggest that the phase state of PEGylated phosphocholine membranes may drastically vary depending on factors such as aliphatic chain length on phosphocholines and PEG-phospholipids, PEG content, and temperature. These findings are summarized in phase drawings and diagrams to demonstrate a striking variety of possible phases. The diagrams can also be instrumental in predicting the phase state of PEGylated phosphocholine membranes, in particular under physiological conditions.

Probing Amphotericin B Single Channel Activity by Membrane Dipole Modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.

Effects of Flavonoids on Dipole Potential of Sterol-Containing Membranes

Flavonoids are hydroxylated polyphenoles from plants which show antioxidant, antiinflammatory, antitumoral, and other therapeutic properties. It is known that flavonoids are membrane-active agents and some of them are able to influence on the membrane structural and dynamic properties. The role of the membrane dipole potential (Vd) is a particular interest due to a powerful impact of Vd on the membrane permeability and lipid-protein interactions. It is known that the flavonoids, phloretin and phloridzin, significantly reduce Vd of phospholipid bilayers [Andersen et al., 1976], but their effects on sterol-containing bilayers are unknown. We estimated the changes of dipole potential (ΔVd) of phosphocholine bilayers containing 33 mol % of cholesterol (the major membrane sterol of mammalian cells) or ergosterol (the major membrane sterol of fungi) after addition into bilayer bathing solution (0.1M KCl pH7.4) of phloretin, phloridzin, genistein, quercetin and biochanin A up to 20 μM. The corresponding calculations were performed assuming that the membrane K+-nonactin steady-state conductance is related to Vd by the Boltzmann distribution [Andersen et al., 1976]. We found that in the presence of phloretin ΔVd of phosphocholine membranes was equal to −130 ± 10 mV, of phosphocholine:cholesterol bilayers is −75 ± 10 mV, and of phosphocholine:ergosterol membranes is −150 ± 10 mV. In contrast to phloretin introduction of phloridzin, genistein, quercetin and biochanin A changes Vd of bilayers of above composition in a similar manner. The average values of ΔVd were −55 ± 10 mV, −40 ± 10 mV, −110 ± 10 mV, and −80 ± 10 mV, respectively. The study was supported in part by RFBR (#09-04-00883), SS-3796.2010.4 and the Program of Presidium of the RAS «Molecular and Cell Biology».

Imaging of blood plasma coagulation at supported lipid membranes

The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO2 and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ∼6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems.

Peptide Adsorption to Lipid Bilayers: Slow Processes Revealed by Linear Dichroism Spectroscopy

The adsorption and insertion kinetics for the association of two 34-residue cyclic peptides with phosphocholine membranes have been studied using circular and linear dichroism approaches. The two peptides studied are identical with the exception of two residues, which are both tyrosine in one of the peptides and tryptophan in the other. Both peptides adopt random coil conformations in solution in the absence of membranes and do not aggregate at concentrations below 20 μM. After addition to liposome dispersions, circular dichroism spectroscopy indicated that both peptides undergo an extremely rapid transformation to a β-conformation that remains unchanged throughout the remainder of the experiment. Linear dichroism (LD) spectroscopy was used to study the kinetics of membrane adsorption and insertion. The data were analyzed by nonlinear least squares approaches, leading to identification of a number of bound states and their corresponding LD spectra. Two pseudo-first order processes could be identified that were common to both peptides. The first occurred with a time constant of the order of 1 min and led to a bound state characterized by weak LD signals, with significant bands corresponding to the transitions of aromatic side chains. The second process occurred with an unusually long time constant of between 75 and 100 min, forming a state with considerably stronger positive LD absorbance in the far-ultraviolet region of the spectrum. For the tyrosine-substituted peptide, a third slow process with a long time constant (76 min) could also be delineated and was attributed to rearrangements of the peptide within the membrane.

Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.

Effect of Tryptophan Oligopeptides on the Size Distribution of POPC Liposomes: A Dynamic Light Scattering and Turbidimetric Study

A chemical regulation of POPC liposome size distribution was investigated, based on the affinity of indole-containing compounds for phosphocholine membranes. In particular, tryptophan oligopeptides have shown interesting properties of size regulation, both when liposomes were formed in their presence and when the peptides were added to a preformed liposome suspension. Combining dynamic light scattering (DLS) and turbidimetric data, it was possible to show how such peptides had an influence on the size distribution of spontaneously formed liposomes prepared by the thin film hydration, reverse-phase evaporation and ethanol (or methanol) injection methods. In the presence of Trp-Trp or Trp-Trp-Trp, a disappearance of large vesicle aggregates was observed, as suggested also by light microscopy analysis. On the contrary, no effect was detected using extruded vesicles. Turbidimetric titration allowed the determination of the relative efficacy of the size regulators, Trp-Trp-Trp being about 20 times more powerful than the dimer, while the monomer had no effect. In addition, other indole-containing compounds and the antimicrobial peptide indolicidin were tested, showing similar behaviours. Discussing the results according to the current knowledge about the preference of Trp residues for interfacial regions in lecithin bilayers, this study confirms the relevant role of tryptophan in the biomembrane binding properties of many peptides and introduces a new behavior in the field of liposomes-peptides interactions.

A calorimetric study of phosphocholine membranes mixed with desmopressin and its diacylated prodrug derivative (DPP)

The influence of the water-soluble peptide, desmopressin (DDAVP) and its dipalmitoylated prodrug derivative (DPP) on the thermal behaviour of three different saturated phosphatidylcholine lipid membranes was investigated by differential scanning calorimetry. For lipid membranes composed of dimyristoyl, dipalmitoyl and distearoyl phosphatidylcholines the addition of DDAVP at concentrations of up to 10 mol% resulted in an insignificant change in the thermodynamic phase behaviour. In contrast, the dipalmitoylated DPP prodrug caused major changes on the lipid membrane phase behaviour manifested as a drastic decrease in the heat capacity peak height and a concomitant broadening of the main phase transition as well as a decrease in the transition enthalpy. In addition, the main phase transition temperature was slightly decreased and the pre-transition of the three phosphatidylcholines was abolished when DPP was present.