Pure POPC Membranes Research Articles

EzAlign: A Tool for Converting Coarse-Grained Molecular Dynamics Structures to Atomistic Resolution for Multiscale Modeling.

Soft condensed matter is challenging to study due to the vast time and length scales that are necessary to accurately represent complex systems and capture their underlying physics. Multiscale simulations are necessary to study processes that have disparate time and/or length scales, which abound throughout biology and other complex systems. Herein we present ezAlign, an open-source software for converting coarse-grained molecular dynamics structures to atomistic representation, allowing multiscale modeling of biomolecular systems. The ezAlign v1.1 software package is publicly available for download at github.com/LLNL/ezAlign. Its underlying methodology is based on a simple alignment of an atomistic template molecule, followed by position-restraint energy minimization, which forces the atomistic molecule to adopt a conformation consistent with the coarse-grained molecule. The molecules are then combined, solvated, minimized, and equilibrated with position restraints. Validation of the process was conducted on a pure POPC membrane and compared with other popular methods to construct atomistic membranes. Additional examples, including surfactant self-assembly, membrane proteins, and more complex bacterial and human plasma membrane models, are also presented. By providing these examples, parameter files, code, and an easy-to-follow recipe to add new molecules, this work will aid future multiscale modeling efforts.

Membrane cholesterol modulates the dynamics and depth of penetration of κ-casein

Aggregation of intrinsically disordered proteins (IDPs) is often triggered by their interaction with membranes, leading to several neurodegenerative diseases such as Parkinson’s, Alzheimer’s, Huntington, prion diseases and spinocerebellar ataxia. This calls for the detailed scrutiny of membrane interaction of IDPs with a special weightage on the intricate role of lipid composition on the protein dynamics. Generally, IDPs lack a well-ordered structure, however, capable of carrying out multiple biological functions because of their higher structural dynamism. In this work, we have chosen an IDP, κ-casein to evaluate its interaction with membranes with varying cholesterol content. Cholesterol plays a vital role in biology because of its involvement in various pathophysiological conditions and differential distribution in organelle membranes. We have deployed steady-state and time-resolved fluorescence spectroscopy to evaluate the dynamics and depth of penetration of κ-casein in the membrane with varying cholesterol composition. Our results demonstrate that the membrane cholesterol influences the depth of penetration and dynamics of κ-casein in the membrane. Taken together, our results reveal that κ-casein presumes higher order of tertiary structure in pure POPC membranes, and tryptophan is located at closer proximity nearer to the location of TMA-DPH (about 10.9 Å from the center of the bilayer). The tertiary structure of κ-casein loses rigidity in membranes containing cholesterol; however, the conformational rigidity is more than what it is in aqueous buffer. In short, we conclude steady-state and time-resolved fluorescence spectroscopy provides an exhaustive information on membrane-IDP interaction.

Effects of lipid heterogeneity on model human brain lipid membranes.

Cell membranes naturally contain a heterogeneous lipid distribution. However, homogeneous bilayers are commonly preferred and utilised in computer simulations due to their relative simplicity, and the availability of lipid force field parameters. Recently, experimental lipidomics data for the human brain cell membranes under healthy and Alzheimer's disease (AD) conditions were investigated, since disruption to the lipid composition has been implicated in neurodegenerative disorders, including AD [R. B. Chan et al., J. Biol. Chem., 2012, 287, 2678-2688]. In order to observe the effects of lipid complexity on the various bilayer properties, molecular dynamics simulations were used to study four membranes with increasing heterogeneity: a pure POPC membrane, a POPC and cholesterol membrane in a 1 : 1 ratio (POPC-CHOL), and to our knowledge, the first realistic models of a healthy brain membrane and an Alzheimer's diseased brain membrane. Numerous structural, interfacial, and dynamical properties, including the area per lipid, interdigitation, dipole potential, and lateral diffusion of the two simple models, POPC and POPC-CHOL, were analysed and compared to those of the complex brain models consisting of 27 lipid components. As the membranes gain heterogeneity, a number of alterations were found in the structural and dynamical properties, and more significant differences were observed in the lateral diffusion. Additionally, we observed snorkeling behaviour of the lipid tails that may play a role in the permeation of small molecules across biological membranes. In this work, atomistic description of realistic brain membrane models is provided, which can add insight towards the permeability and transport pathways of small molecules across these membrane barriers.

The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry.

We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.

Determinants of Orexin Receptor Binding and Activation-A Molecular Dynamics Study.

We assess the stability of two previously suggested binding modes for the neuropeptide orexin-A in the OX2 receptor through extensive molecular dynamics simulations. As the activation determinants of the receptor remain unknown, we simulated an unliganded receptor and two small-molecular ligands, the antagonist suvorexant and the agonist Nag26 for comparison. Each system was simulated in pure POPC membrane as well as in the 25% cholesterol–POPC membrane. In total, we carried out 36 μs of simulations. Through this set of simulations, we report a stable binding mode for the C-terminus of orexin-A. In addition, we suggest interactions that would promote orexin receptor activation, as well as others that would stabilize the inactive state.

Effect of ubiquinone-10 on the stability of biomimetic membranes of relevance for the inner mitochondrial membrane

Ubiquinone-10 (Q10) plays a pivotal role as electron-carrier in the mitochondrial respiratory chain, and is also well known for its powerful antioxidant properties. Recent findings suggest moreover that Q10 could have an important membrane stabilizing function. In line with this, we showed in a previous study that Q10 decreases the permeability to carboxyfluorescein (CF) and increases the mechanical strength of 1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) membranes. In the current study we report on the effects exerted by Q10 in membranes having a more complex lipid composition designed to mimic that of the inner mitochondrial membrane (IMM). Results from DPH fluorescence anisotropy and permeability measurements, as well as investigations probing the interaction of liposomes with silica surfaces, corroborate a membrane stabilizing effect of Q10 also in the IMM-mimicking membranes. Comparative investigations examining the effect of Q10 and the polyisoprenoid alcohol solanesol on the IMM model and on membranes composed of individual IMM components suggest, moreover, that Q10 improves the membrane barrier properties via different mechanisms depending on the lipid composition of the membrane. Thus, whereas Q10's inhibitory effect on CF release from pure POPC membranes appears to be directly and solely related to Q10's lipid ordering and condensing effect, a mechanism linked to Q10's ability to amplify intrinsic curvature elastic stress dominates in case of membranes containing high proportions of palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 1. Lipid Dynamics

Properties of biomembranes are modified in presence of additives such as cholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a photoactive variety of chol since its azo-headgroup exhibits a reversible change of the conformation upon illumination (365nm: trans to cis; 455nm: cis to trans). Quasielastic neutron scattering (QENS) was applied to resolve lipid dynamics within biomimetic membranes consisting of a 1-palmitoyl-2-oleoyl-sn-phosphatidyl-choline (POPC) matrix with embedded azo-chol in either of the two conformational states. A pure POPC membrane and also a POPC membrane with inactive chol were used as control systems. The results report on the fast intramolecular motion of the protons in the POPC chains, a slow hop diffusion and an even slower mode both of the whole POPC molecule. The intramolecular motion and the hop diffusion are modified by the presence of azo-chol in the host membrane as compared with the control systems, but do not depend on the isomerization state. The slowest mode is though sensitive to the isomerization. The findings show the capacity of azo-chol for tuning the dynamics and most probably also the structures of the host lipid membrane though photoactivation.

Interaction of HIV-1 Gag Protein's Ma Membrane Binding Domain with Membrane Mimics Probed by Low- and Wide-Angle X-Ray Scattering

The structural Gag protein from the HIV-1 virus is required for assembling new virus particles within an infected T-cell; Gag's binding to the inner leaflet of the host plasma membrane is the first step in this process. In an effort to understand the molecular and energetic details of this binding, we studied the N-terminal 31 amino acids of Gag's MA membrane binding domain with lipid membrane mimics: PC, PC:PE, PC:PS, PC:PI, PC:PIP, PC:PIP2, PC:PE:PI, PC:PE:PIP, PC:PE:PIP2, PC:PE:PIP2:cholesterol and PC:PE:PS:cholesterol in various molar ratios. Oriented, fully hydrated lipid mimics with/without the myristoylated (MAmyr) and non-myristolyated (MA) peptide were X-rayed at the Cornell High Energy Synchrotron Source. We found that MAmyr lowered KC (softened) pure POPC membranes more than did MA; in general, both peptides lowered KC for all 18 mimics. MAmyr increased slightly Sxray (chain order) of PS-containing membrane mimics, but both peptides decreased Sxray slightly with increasing concentrations of PI, PIP or PIP2 when mixed with POPC. When POPC:POPE was mixed with PI, PIP or PIP2 (5:3:2), PI decreased, PIP had no effect and PIP2 increased chain order with either peptide compared to controls. The head-to-head spacing (DHH) was decreased by both peptides (1-3 A) in most mimics. In pure POPC, or when PI, PIP or PIP2 was mixed with POPC, modeling suggested a penetration of MAmyr into the hydrocarbon region, compared to an interfacial headgroup position for MA. Therefore the effect of MA peptides on membrane structure and properties depends on the composition of the lipid mimics as well as on the myristoyl group.Acknowledgments: NIH R01 GM44976 (STN and JFN).

Sensing Small Molecule Interactions with Lipid Membranes by Local pH Modulation

Herein, we utilized a label-free sensing platform based on pH modulation to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic, and planar supported lipid bilayers (SLBs). The SLBs were patterned inside a flow cell, allowing for various concentrations of tetracaine to be introduced over the surface in a buffer solution. Studies with membranes containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180 ± 47 μm for this small molecule-membrane interaction. Adding cholesterol to the SLBs decreased the affinity between tetracaine and the bilayers, while this interaction tightened when POPE (1-hexadecanoyl-2-(9-Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) was added. Studies were also conducted with three negatively charged membrane lipids, POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)), and ganglioside GM1. All three measurements gave rise to a similar tightening of the apparent Kd value compared with pure POPC membranes. The lack of chemical specificity with the identity of the negatively charged lipid indicated that the tightening was largely electrostatic. Through a direct comparison with ITC measurements, it was found that the pH modulation sensor platform offers a facile, inexpensive, highly sensitive, and rapid method for the detection of interactions between putative drug candidates and lipid bilayers. As such, this technique may potentially be exploited as a screen for drug development and analysis.

Biophysical Characterization of Peptide Membrane Interactions and Membrane Permeabilization

Certain membrane-active amphipathic peptides, such as antimicrobial peptides, possess a characteristic feature of having several cationic amino acid residues at either N- or C-terminus. In an attempt to determine the functional significance of C-terminal cationic residues, we have studied the membrane binding mode and membrane pore formation by a 20-amino acid synthetic amphipathic peptide. Two cationic residues were replaced to Leu or Glu to see how the membrane binding and permeabilization activities of the peptide are modulated by charge neutralization and charge reversal mutations. The depth of membrane insertion of the wild type and mutant peptides was determined by measuring the quenching of the fluorescence of a single, C-terminally located tryptophan by dibromo-phosphatidylcholines brominated at various positions of the acyl chains, using pure POPC and 70% POPC + 30% POPG in lipid vesicles. In case of pure POPC membranes, Trp in all three peptides was located at around 9 Å from the membrane center, while for 30% POPG, Trp was inserted much deeper. Studies on peptide-mediated calcein release from phospholipid vesicles were conducted to see if the peptides are able to cause membrane pore formation and content release. Peptides with positively charged residues showed maximum calcein release for both lipid compositions whereas negative or hydrophobic residues were less effective in vesicle permeabilization. Preliminary ATR-FTIR experiments using both POPC and POPG have been performed to identify the orientation of the peptides within the lipid bilayer and their effect of the lipid order.

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.

Phase coexistence in a triolein–phosphatidylcholine system. Implications for lysosomal membrane properties

The effects of tri- and monoglycerides on phospholipid (POPC) membranes were studied using spectroscopical methods. Triolein was found to form two types of POPC-rich membranes, both with POPC or as a three-component system with monopalmitin. These two membrane types were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. Marked differences were seen in the physical properties of these phases, even though only minor compositional variation was detected. The light, less polar phase was found to be less ordered and more fluid and seemed to allow significantly lower amount of water penetration into the membrane–water interface than pure POPC membrane. The heavy phase, apart from their slightly altered water penetration, resembled more a pure POPC membrane. As triglycerides are present in lysosomal membranes, the present results can be seen as an implication for polarity-based water permeability barrier possibly contributing to the integrity of lysosomes.

Fuscopeptins, antimicrobial lipodepsipeptides from Pseudomonas fuscovaginae, are channel forming peptides active on biological and model membranes

FP-A and FP-B are LDPs produced by the plant pathogen Pseudomonas fuscovaginae. As expected from their primary structure, they shared a similar mechanism of action with the better characterized SPs, synthesized by strains of Pseudomonas syringae pv. syringae. Indeed, they displayed hemolytic activity on human erythrocytes and were able to induce calcein release from LUVs: the effect was dependent on the concentration of the FPs and the lipid composition of the liposome and, in particular, it increased with the SM content of the membrane. The permeabilizing activity was further investigated on PLMs. FPs were able to open pores on pure POPC membranes. Pore opening was strongly voltage dependent: by switching the potential from negative to positive values, an increase in the absolute amplitude of transmembrane current was induced with simultaneous closure of pores. In 0.1 M KCl both FPs' pores had a conductance of 4 and 9 pS at - 140 mV and + 140 mV, respectively. Studies on ion selectivity indicated that FPs formed cation-selective channels.

Physical properties of the lipid bilayer membrane made of calf lens lipids: EPR spin labeling studies

The physical properties of a membrane derived from the total lipids of a calf lens were investigated using EPR spin labeling and were compared with the properties of membranes made of an equimolar 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol (POPC/Chol) mixture and of pure POPC. Conventional EPR spectra and saturation–recovery curves show that spin labels detect a single homogenous environment in all three membranes. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are practically identical in lens lipid and POPC/Chol membranes, but differ drastically from profiles in pure POPC membranes. In both lens lipid and POPC/Chol membranes, the lipids are strongly immobilized at all depths, which is in contrast to the high fluidity of the POPC membrane. Hydrophobicity and oxygen transport parameter profiles in lens lipid and POPC/Chol membranes have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. At this position, hydrophobicity increases from the level of methanol to the level of hexane, and the oxygen transport parameter increases by a factor of 2–3. These profiles in POPC membranes are bell-shaped. It is concluded that the high level of cholesterol in lens lipids makes the membrane stable, immobile, and impermeable to both polar and nonpolar molecules.

The Sensitivity of Lipid Domains to Small Perturbations Demonstrated by the Effect of Triton

The hypothesis of lipid rafts describes functional domains in biological membranes. It is often assumed that rafts form by spontaneous de-mixing of certain lipids and that they can be isolated as detergent-resistant membrane particles (DRMs) using the detergent Triton X-100 (TX). Here, we present a model that describes the process of domain formation in membranes in the presence and in the absence of TX. We measure the interactions between TX and an equimolar mixture of sphingomyelin (SM), cholesterol (Cho), and 1-palmitoyl-2-oleoyl-3-sn-glycero-phosphatidylcholine (POPC) (1:1:1, mol) by means of isothermal titration calorimetry. Comparison with pure POPC membranes reveals a very unfavorable interaction between TX and SM/Cho, which causes a substantial tendency to segregate these molecules into separate, DRM-like (SM-rich) and fluid (TX-rich), domains. If rafts are indeed formed by spontaneous de-mixing of PC and SM/Cho, they must be very sensitive, and perturbations caused by techniques used to study rafts could lead to misleading results. If, however, rafts are much more stable than PC–SM–Cho domains, there must be an unknown raft stabilizer. Subtle changes of such a promoter could serve to modulate raft function.