Architecture of bacterial lipid A in solution. A neutron small-angle scattering study

The phase structure of isolated bacterial lipid A, the lipid anchor of the lipopolysaccharides of the outer membrane of Gram-negative bacteria, has been investigated by neutron small-angle scattering. The shape of the scattering curves obtained at different H2O/2H2O ratios revealed a lamellar organisation of the lipid A at neutral pH both above and below its main phase temperature (approximately 40-45 degrees C). Analysis of the scattering curves and interpretation of the corresponding thickness distance distribution functions of the lamellar aggregates led to a model in which the lipid A molecules form a bilayer of about 5 nm in thickness. This value for the thickness of the bilayer, as well as the neutron-scattering density profile across the bilayer, can be explained by a molecular model which shows interdigitation of the fatty acid chains of the lipid A.     

The water permeability of the monoolein/triolein bilayer membrane

The water permeability of the lipid bilayer can be used as a probe of membrane structure. A simple model of the bilayer, the liquid hydrocarbon model, views the membrane as a thin slice of bulk hydrocarbon liquid. A previous study (Petersen, D. (1980) Biochim. Biophys. Acta 600, 666–677) showed that this model does not accurately predict the water permeability of the monoolein/n-hexadecane bilayer: the measured activation energy for water permeation is 50% above the predicted value. From this it was inferred that the hydrocarbon chains in the lipid bilayer are more ordered than in the bulk hydrocarbon liquid. The present study tests the liquid hydrocarbon model for the monoolein/triolein bilayer, which has been shown to contain very little triolein in the plane of the membrane (Waldbillig, R.C. and Szabo, G. (1979) Biochim. Biophys. Acta 557, 295–305). Measurements of the water permeability coefficient of the bilayer are compared with predictions of the liquid hydrocarbon model based on measurements of the water permeability coefficient of bulk 8-heptadecene. The predicted and measured values agree quite closely over the temperature range studied (15–35°C): the predicted activation energy is 11.1±0.2 kcal/mol, whereas the measured activation energy for the bilayer is 9.8±0.7 kcal/mol. This close agreement is in contrast with the monoolein/n-hexadecane results and suggests that, insofar as water permeation is concerned, the liquid hydrocarbon model quite closely represents the monoolein/triolein bilayer.     

Penetration of Lysozyme and Cytochrome C in Lipid Bilayer: Fluorescent Study

Lysozyme and cytochrome c (CytC) are well-investigated proteins. Their specific interactions with lipid membranes, however, keep surprising secrets. Lysozyme destroys bacterial membrane; CytC binds hydrophobically to alkyl chains of the membrane lipid tails, indicating that both proteins are able to interact directly with the inner membrane components, especially with the fatty acyl chains of membrane lipids. The degrees of integration, depth of localization in the hydrophobic interior of different types of model membranes, and the type of interaction of lysozyme and CytC with surrounding lipids were investigated by fluorescent spectroscopy. Three different fluorescent markers, located at approximately 6.5, 9, and 18 Å into the lipid bilayer, were used. In addition, liposomes were designed as electrically neutral or positively or negatively charged to unravel the importance of the net electrical charge for lipid/protein interaction. CytC penetrates deeper into the lipid bilayer in comparison with lysozyme, and data are discussed in the terms of Stern–Volmer quenching of fluorescence.     

The Role of the Lipid Bilayer in Tau Aggregation

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the β-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.     

Transmembrane Lipid Migration in Planar Asymmetric Bilayer Membranes

Planar asymmetric bilayer membranes, formed by apposing a monolayer of the neutral lipid glyceroldioleate (GDO) with one of the negatively charged lipid oleyl acid phosphate (OAP), were used to measure the rate of transmembrane OAP migration. The assay for this lipid flip-flop was the interaction of Ca²⁺ ions with negatively charged lipids which causes membranes to break: when Ca²⁺ is added to the compartment limited initially by the neutral lipid, flip-flop of the charged lipid eventually results in membrane breakdown. At 22 ± 2°C, in the absence of an externally applied electric field, an upper limit to the half time of OAP flip-flop was measured as 18.7 h, with a tentative lower limit of 14.4 h.     

Propagating Subterahertz Longitudinal Phonon-like Modes in a Lipid Bilayer in Aqueous Medium

It has been revealed recently that the subterahertz/terahertz vibrational motions in enzymes and DNA immersed in aqueous solutions can be underdamped. Importantly, these motions are associated with coherent delocalized modes that control functional processes. Analogous propagating phonon-like modes have been found in free hydrated lipid bilayers. In the present work, subterahertz (frequencies of the order of tens and hundreds of gigahertz) longitudinal acoustic oscillations in a bilayer lipid membrane immersed in aqueous medium are investigated theoretically. We consider driven oscillations excited by tangential mechanical tensions at the bilayer surfaces and thermally induced phonon modes. The analysis is based on: (i) a generalized hydrodynamic model of two-dimensional lipid bilayer in aqueous medium; (ii) known estimates of frequencies and lifetimes of longitudinal acoustic phonons in free hydrated lipid bilayer and in water, which were obtained in the experiments on non-elastic X-ray scattering and the molecular dynamics simulations. We show that the membrane phonon-like excitations are underdamped for the typical values of the system parameters, and the contribution of aqueous medium to the membrane mode damping is small compared to the contribution of the lipid bilayer. The obtained results suggest the possibility of realization of thermally induced longitudinal membrane phonons in physiological conditions, as well as the possibility of resonance amplification of the impact of subnanosecond electric impulses and impulses of subterahertz electromagnetic radiation on membrane dynamics.     

Lipid Clustering Correlates with Membrane Curvature as Revealed by Molecular Simulations of Complex Lipid Bilayers

Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP₂), in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP₂, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side) regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.     

Molecular dynamics simulations of local anesthetic articaine in a lipid bilayer

In order to investigate structural and dynamical properties of local anesthetic articaine in a model lipid bilayer, a series of molecular dynamics simulations have been performed. Simulations were carried out for neutral and charged (protonated) forms of articaine inserted in fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. For comparison purpose, a fully hydrated DMPC bilayer without articaine was also simulated. The length of each simulation was 200 ns. Various properties of the lipid bilayer systems in the presence of both charged and uncharged forms of articaine taken at two different concentrations have been examined: membrane area per lipid, mass density distributions, order parameters, radial distribution functions, head group tilt, diffusion coefficients, electrostatic potential, etc, and compared with results of previous simulations of DMPC bilayer in the presence of lidocaine. It was shown that addition of both charged and neutral forms of articaine causes increase of the dipole electrostatic potential in the membrane interior.     

Properties of Hexadecaprenyl Monophosphate/Dioleoylphosphatidylcholine Vesicular Lipid Bilayers

In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl monophosphate (C₈₀-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques. The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C₈₀-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined. Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation energy and the membrane breakdown voltage for the various value of C₈₀-P/DOPC mole ratio, respectively. The TEM micrographs of C₈₀-P, DOPC and C₈₀-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the next step in understanding the origin of protocells. Received: 10 January 2000/Revised: 7 June 2000     

Amyloid Aggregation on Lipid Bilayers and Its Impact on Membrane Permeability

Fibrillar protein aggregates (amyloids) are involved in several common pathologies, e.g., Alzheimer's disease and type II diabetes. Accumulating evidence suggests that toxicity in amyloid-related diseases originates from the deposition of protein aggregates on the cell membrane, which results in bilayer disruption and cell leakage. The molecular mechanism of damage to the membrane, however, is still obscure. To shed light on it we have performed coarse-grained molecular dynamics simulations of fibril-forming amphipathic peptides in the presence of lipid vesicles. The simulation results show that highly amyloidogenic peptides fibrillate on the surface of the vesicle, damaging the bilayer and promoting leakage. In contrast, the ordered aggregation of peptides with low amyloidogenicity is hindered by the vesicles. Remarkably, leakage from the vesicle is caused by growing aggregates, but not mature fibrils. The simulation results provide a basis for understanding the range of aggregation behavior that is observed in experiments with fibril-forming (poly)peptides.     

Erythrocyte Membrane Model with Explicit Description of the Lipid Bilayer and the Spectrin Network

The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do not impose a complete attachment of the filaments to the lipid bilayer.     

Lipopolysaccharide-Induced Dynamic Lipid Membrane Reorganization: Tubules,Perforations, and Stacks

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na⁺ leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca²⁺ gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions.     

Lipopolysaccharide-Induced Dynamic Lipid Membrane Reorganization: Tubules,Perforations, and Stacks

Lipopolysaccharide (LPS) is a unique lipoglycan, with two major physiological roles: 1), as a major structural component of the outer membrane of Gram-negative bacteria and 2), as a highly potent mammalian toxin when released from cells into solution (endotoxin). LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain, leaving the hydrophilic polysaccharide chain exposed to the exterior polar solvent. Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of monovalent cations forms highly mobile negatively-charged aggregates. Yet, much of our understanding of LPS and its interactions with the cell membrane does not take into account its amphiphilic biochemistry and charge polarization. Herein, we report fluorescence microscopy and atomic force microscopy analysis of the interaction between LPS and fluid-phase supported lipid bilayer assemblies (sLBAs), as model membranes. Depending on cation availability, LPS induces three remarkably different effects on simple sLBAs. Net-negative LPS-Na⁺ leads to the formation of 100-μm-long flexible lipid tubules from surface-associated lipid vesicles and the destabilization of the sLBA resulting in micron-size hole formation. Neutral LPS-Ca²⁺ gives rise to 100-μm-wide single- or multilamellar planar sheets of lipid and LPS formed from surface-associated lipid vesicles. Our findings have important implications about the physical interactions between LPS and lipids and demonstrate that sLBAs can be useful platforms to study the interactions of amphiphilic virulence factors with cell membranes. Additionally, our study supports the general phenomenon that lipids with highly charged or bulky headgroups can promote highly curved membrane architectures due to electrostatic and/or steric repulsions.     

Erythrocyte Membrane Model with Explicit Description of the Lipid Bilayer and the Spectrin Network

The membrane of the red blood cell (RBC) consists of spectrin tetramers connected at actin junctional complexes, forming a two-dimensional (2D) sixfold triangular network anchored to the lipid bilayer. Better understanding of the erythrocyte mechanics in hereditary blood disorders such as spherocytosis, elliptocytosis, and especially, sickle cell disease requires the development of a detailed membrane model. In this study, we introduce a mesoscale implicit-solvent coarse-grained molecular dynamics (CGMD) model of the erythrocyte membrane that explicitly describes the phospholipid bilayer and the cytoskeleton, by extending a previously developed two-component RBC membrane model. We show that the proposed model represents RBC membrane with the appropriate bending stiffness and shear modulus. The timescale and self-consistency of the model are established by comparing our results with experimentally measured viscosity and thermal fluctuations of the RBC membrane. Furthermore, we measure the pressure exerted by the cytoskeleton on the lipid bilayer. We find that defects at the anchoring points of the cytoskeleton to the lipid bilayer (as in spherocytes) cause a reduction in the pressure compared with an intact membrane, whereas defects in the dimer-dimer association of a spectrin filament (as in elliptocytes) cause an even larger decrease in the pressure. We conjecture that this finding may explain why the experimentally measured diffusion coefficients of band-3 proteins are higher in elliptocytes than in spherocytes, and higher than in normal RBCs. Finally, we study the effects that possible attractive forces between the spectrin filaments and the lipid bilayer have on the pressure applied on the lipid bilayer by the filaments. We discover that the attractive forces cause an increase in the pressure as they diminish the effect of membrane protein defects. As this finding contradicts with experimental results, we conclude that the attractive forces are moderate and do not impose a complete attachment of the filaments to the lipid bilayer.     

Peptide Aggregation and Pore Formation in a Lipid Bilayer: A Combined Coarse-Grained and All Atom Molecular Dynamics Study

We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by alamethicin peptides in a hydrated lipid bilayer. In the 1-μs CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 μs is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the α-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of alamethicin oligomers formed by perfectly aligned helical alamethicin peptides in a lipid bilayer.     

Fluorinated amphiphiles control the insertion of α-hemolysin pores into lipid bilayers

The insertion of fully folded and assembled ion channels and pores into planar lipid bilayers for electrical recording has been facilitated by the use of conventional detergents at a final concentration below the critical micelle concentration (CMC). After the desired number of channels or pores (often one) has been incorporated into a bilayer, it is important to prevent further insertion events, which is often done by awkward techniques such as perfusion. Here, we show that the addition of single-chain fluorinated amphiphiles (F-amphiphiles) with zwitterionic, simple neutral, and neutral oligomeric headgroups at a concentration above the CMC prevents the further insertion of staphylococcal α-hemolysin pores, MspA pores, and Kcv potassium channels into lipid bilayers. We found the commercially available F(6)FC (fluorinated fos-choline with a C(6)F(13)C(2)H(4) chain) to be the least perturbing and most effective agent for this purpose. Bilayers are known to be resistant to F-amphiphiles, which in this case we suppose sequester the pores and channels within amphiphile aggregates. We suggest that F-amphiphiles might be useful in the fabrication of bilayer arrays for nanopore sensor devices and the rapid screening of membrane proteins.     

Chemical versus Mechanical Perturbations on the Protonation State of Arginine in Complex Lipid Membranes: Insights from Microscopic pKa Calculations

Charged amino acids such as Arginine play important roles in many membrane-mediated biological processes such as voltage gating of ion channels and membrane translocation of cell penetration peptides. It is well established that local membrane deformation and formation of water defects are crucial to the stabilization of charged species in contact with the membrane, which suggests that mechanical properties of the membrane are relevant although a clear connection has not been established. As a quantitative measure, we study how changes in the composition and therefore mechanical properties of a lipid bilayer influence the pK_a of Arg in the membrane center using free energy simulations. Compared to previous studies in a single-component lipid bilayer containing saturated lipids or lipids with a modest degree of unsaturation, substantially larger pK_a shifts are observed in the presence of highly unsaturated lipid tails and cholesterol. Moreover, the underlying molecular mechanisms for the pK_a perturbation are distinct in different systems, with the unsaturated lipid tails mainly destabilizing the charged state of Arg and the cholesterol stabilizing the neutral state of Arg. The observed behaviors in both cases are at odds with predictions based on mechanical considerations at a mesoscopic level—highlighting that, while mechanical considerations are useful for stimulating hypothesis, their applicability to dissecting phenomena at the molecular-length scale is rather limited.     

An Asymmetry in Monolayer Tension Regulates Lipid Droplet Budding Direction

Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the endoplasmic reticulum intermonolayer space. The lipids next demix to form lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol. This directional LD formation is critical to energy metabolism, but its mechanism remains poorly understood. Here, we reconstituted the LD formation topology by embedding artificial LDs into the intermonolayer space of bilayer vesicles. We provide experimental evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, dictated by the equilibrium of surface tensions. We thereupon determined that slight tension asymmetries between the membrane monolayers regulate the droplet budding side. A differential regulation of lipid or protein composition around a forming LD can generate a monolayer tension asymmetry that will determine the LD budding side. Our results offer, to our knowledge, new insights on how the proteins might regulate LD formation side by generating a monolayer tension asymmetry.     

Atomistic-level study of the interactions between hIAPP protofibrils and membranes: Influence of pH and lipid composition

The pathology of type 2 diabetes mellitus is associated with the aggregation of human islet amyloid polypeptide (hIAPP) and aggregation-mediated membrane disruption. The interactions of hIAPP aggregates with lipid membrane, as well as the effects of pH and lipid composition at the atomic level, remain elusive. Herein, using molecular dynamics simulations, we investigate the interactions of hIAPP protofibrillar oligomers with lipids, and the membrane perturbation that they induce, when they are partially inserted in an anionic dipalmitoyl-phosphatidylglycerol (DPPG) membrane or a mixed dipalmitoyl-phosphatidylcholine (DPPC)/DPPG (7:3) lipid bilayer under acidic/neutral pH conditions. We observed that the tilt angles and insertion depths of the hIAPP protofibril are strongly correlated with the pH and lipid composition. At neutral pH, the tilt angle and insertion depth of hIAPP protofibrils at a DPPG bilayer reach ~52° and ~1.62 nm with respect to the membrane surface, while they become ~77° and ~1.75 nm at a mixed DPPC/DPPG membrane. The calculated tilt angle of hIAPP at DPPG membrane is consistent with a recent chiral sum frequency generation spectroscopic study. The acidic pH induces a smaller tilt angle of ~40° and a shallower insertion depth (~1.24 nm) of hIAPP at the DPPG membrane surface, mainly due to protonation of His18 near the turn region. These differences mainly result from a combination of distinct electrostatic, van der Waals, hydrogen bonding and salt-bridge interactions between hIAPP and lipid bilayers. The hIAPP-membrane interaction energy analysis reveals that besides charged residues K1, R11 and H18, aromatic residues Phe15 and Phe23 also exhibit strong interactions with lipid bilayers, revealing the crucial role of aromatic residues in stabilizing the membrane-bound hIAPP protofibrils. hIAPP-membrane interactions disturb the lipid ordering and the local bilayer thickness around the peptides. Our results provide atomic-level information of membrane interaction of hIAPP protofibrils, revealing pH-dependent and membrane-modulated hIAPP aggregation at the early stage.     

Small-Molecule Photostabilizing Agents are Modifiers of Lipid Bilayer Properties

Small-molecule photostabilizing or protective agents (PAs) provide essential support for the stability demands on fluorescent dyes in single-molecule spectroscopy and fluorescence microscopy. These agents are employed also in studies of cell membranes and model systems mimicking lipid bilayer environments, but there is little information about their possible effects on membrane structure and physical properties. Given the impact of amphipathic small molecules on bilayer properties such as elasticity and intrinsic curvature, we investigated the effects of six commonly used PAs—cyclooctatetraene (COT), para-nitrobenzyl alcohol (NBA), Trolox (TX), 1,4-diazabicyclo[2.2.2]octane (DABCO), para-nitrobenzoic acid (pNBA), and n-propyl gallate (nPG)—on bilayer properties using a gramicidin A (gA)-based fluorescence quench assay to probe for PA-induced changes in the gramicidin monomer↔dimer equilibrium. The experiments were done using fluorophore-loaded large unilamellar vesicles that had been doped with gA, and changes in the gA monomer↔dimer equilibrium were assayed using a gA channel-permeable fluorescence quencher (Tl⁺). Changes in bilayer properties caused by, e.g., PA adsorption at the bilayer/solution interface that alter the equilibrium constant for gA channel formation, and thus the number of conducting gA channels in the large unilamellar vesicle membrane, will be detectable as changes in the rate of Tl⁺ influx—the fluorescence quench rate. Over the experimentally relevant millimolar concentration range, TX, NBA, and pNBA, caused comparable increases in gA channel activity. COT, also in the millimolar range, caused a slight decrease in gA channel activity. nPG increased channel activity at submillimolar concentrations. DABCO did not alter gA activity. Five of the six tested PAs thus alter lipid bilayer properties at experimentally relevant concentrations, which becomes important for the design and analysis of fluorescence studies in cells and model membrane systems. We therefore tested combinations of COT, NBA, and TX; the combinations altered the fluorescence quench rate less than would be predicted assuming their effects on bilayer properties were additive. The combination of equimolar concentrations of COT and NBA caused minimal changes in the fluorescence quench rate.