Molecular dynamics simulations of the human ocular lens with age and cataract

The human ocular lens consists primarily of elongated, static fibers characterized by high stability and low turnover, which differ dramatically in their composition and properties from other biological membranes. Cholesterol (Chol) and sphingolipids (SL) are present at high concentrations, including saturated SLs, such as dihyrosphingomyelin (DHSM). Past molecular dynamics simulations demonstrated that the presence of DHSM and high Chol concentration contributes to higher order in lipid membranes. This current study simulated more complex models of human lens membranes. Models were developed representing physiological compositions in cataractous lenses aged 74 ± 6 years and in healthy lenses aged 22 ± 4, 41 ± 6, and 69 ± 3 years. With older age, Chol and ceramide concentrations increase and glycerophospholipid concentration decreases. With cataract, ceramide concentration increases and Chol and glycerophospholipid concentrations decrease. Surface area per lipid, deuterium order parameters (S_CD), sterol tilt angle, electron density profiles, bilayer thickness, chain interdigitation, two-dimensional radial distribution functions (2D-RDF), lipid clustering, and hydrogen bonding were calculated for all simulations. All systems exhibited low surface area per lipid and high bilayer thickness, indicative of strong vertical packing. S_CD parameters suggest similarly, with saturated tails in the hydrophobic core of the membrane having elevated order. Vertical packing and acyl tail order increased with both age and cataract condition. Lateral diffusion decreased with age and cataracts, with the older and cataractous models demonstrating increased long-range structure by the 2D-RDF analysis. In future work examining the membrane proteins of the lens, these models can serve as a physiologically accurate representation of the lens lipidome.     

Structural divergence among cannabinoids influences membrane dynamics: A H Solid-State NMR analysis

Cannabinoids are compounds that can modulate neuronal functions and immune responses via their activity at the CB₁ receptor. We used ²H NMR order parameters and relaxation rate determination to delineate the behavior of magnetically aligned phospholipid bilayers in the presence of several structurally distinct cannabinoid ligands. THC (Δ⁹-Tetrahydrocannabinol) and WIN-55,212-2 were found to lower the phase transition temperature of the DMPC and to destabilize their acyl chains leading to a lower average S_CD (≈ 0.13), while methanandamide and CP-55,940 exhibited unusual properties within the lipid bilayer resulting in a greater average S_CD (≈ 0.14) at the top of the phospholipid upper chain. The CB₁ antagonist AM281 had average S_CD values that were higher than the pure DMPC lipids, indicating a stabilization of the lipid bilayer. R_1Z versus |S_CD|² plots indicated that the membrane fluidity is increased in the presence of THC and WIN-55,212-2. The interaction of CP-55,940 with a variety of zwitterionic and charged membranes was also assessed. The unusual effect of CP-55,940 was present only in bicelles composed of DMPC. These studies strongly suggest that cannabinoid action on the membrane depends upon membrane composition as well as the structure of the cannabinoid ligands.     

Molecular Dynamics Simulations of the Rotary Motor F0 under External Electric Fields across the Membrane

The membrane-bound component F₀, which is a major component of the F₀F₁-ATP synthase, works as a rotary motor and plays a central role in driving the F₁ component to transform chemiosmotic energy into ATP synthesis. We conducted molecular dynamics simulations of b₂-free F₀ in a 1-palmitoyl-2-oleoyl-phosphatidylcholine lipid bilayer for tens of nanoseconds with two different protonation states of the cAsp-61 residue at the interface of the a-c complex in the absence of electric fields and under electric fields of ±0.03 V/nm across the membrane. To our surprise, we observed that the upper half of the N-terminal helix of the c₁ subunit rotated about its axis clockwise by 30°. An energetic analysis revealed that the electrostatic repulsion between this N-terminal helix and subunit c₁₂ was a major contributor to the observed rotation. A correlation map analysis indicated that the correlated motions of residues in the interface of the a-c complex were significantly reduced by external electric fields. The deuterium order parameter (S_CD) profile calculated by averaging all the lipids in the F₀-bound bilayer was not very different from that of the pure bilayer system, in agreement with recent ²H solid-state NMR experiments. However, by delineating the lipid properties according to their vicinity to F₀, we found that the S_CD profiles of different lipid shells were prominently different. Lipids close to F₀ formed a more ordered structure. Similarly, the lateral diffusion of lipids on the membrane surface also followed a shell-dependent behavior. The lipids in the proximity of F₀ exhibited very significantly reduced diffusional motion. The numerical value of S_CD was anticorrelated with that of the diffusion coefficient, i.e., the more ordered lipid structures led to slower lipid diffusion. Our findings will help elucidate the dynamics of F₀ depending on the protonation state and electric field, and may also shed some light on the interactions between the motor F₀ and its surrounding lipids under physiological conditions, which could help to rationalize its extraordinary energy conversion efficiency.     

Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?

In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (P_M) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the P_M across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the P_M of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~ 10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.     

Lipids, curvature stress, and the action of lipid prodrugs: free fatty acids and lysolipid enhancement of drug transport across liposomal membranes

Molecular shape and its impact on bilayer curvature stress are powerful concepts for describing the effects of lipids and fatty acids on fundamental membrane properties, such as passive permeability and derived properties like drug transport across liposomal membranes. We illustrate these relationships by studying the effects of fatty acids and lysolipids on the permeation of a potent anti-cancer drug, doxorubicin, across the bilayer of a liposome in which the drug is encapsulated. Using a simple fluorescence assay, we have systematically studied the passive permeation of doxorubicin across liposomal membranes in different lipid phases: the solid-ordered phase (DPPC bilayers), the liquid-disordered phase (POPC lipid bilayers), and the liquid-ordered phase induced by high levels of cholesterol (DOPC + cholesterol lipid bilayers). The effect of different free fatty acids (FA) and lysolipids (LL), separately and in combination, on permeability was assessed to elucidate the possible mechanism of phospholipase A₂-triggered release in cancer tissue of liposomal doxorubicin formulations. In all cases, FAs applied separately lead to significant enhancement of permeability, most pronounced in liquid-disordered bilayers and less pronounced in solid and solid-ordered bilayers. LLs applied separately had only a marginal effect on permeability. FA and LL applied in combination lead to a synergistic enhancement of permeability in solid bilayers, whereas in liquid-disordered bilayers, the combined effect suppressed the otherwise strong permeability enhancement due to the FAs.     

Interaction of enantiomeric alkylacyl and diacylglycerophosphocholines with cholesterol in bilayer membranes

The sn-1 and sn-3 isomers of dioleoylglycerophosphocholine form vesicles of the same size as the racemic lipid. Identical permeability coefficients were found for the diffusion of glucose and chloride across bilayer membranes of vesicles consisting of these lipids. Vesicles made of mixtures of enantiomeric or racemic dioleoyllecithin with 30 mol% cholesterol have identical radii. Cholesterol reduces the permeability of bilayers for glucose and chloride irrespective of the steric configuration of the constituent phospholipid. Increasing concentrations of cholesterol (17, 33 and 50 mol%, respectively) broaden the (CH₂)_n signal in the ¹H-NMR-spectra (90 MHz) of unilamellar vesicles containing sn-1, sn-3 or rac alkyloleoylglycerophosphocholine to the same extent. These results indicate that the steric configuration of phospholipids has no gross effect on the arrangement of phospholipids and cholesterol in bilayer membranes.     

Liquid-liquid phase transition temperatures increase when lipid bilayers are supported on glass

Membranes made from certain ternary mixtures of lipids can display coexisting liquid phases. In giant unilamellar vesicles, these phases appear as liquid domains which diffuse and coalesce after the vesicle is cooled below its miscibility transition temperature (T_m). Converting vesicles to supported lipid bilayers alters the mobility of the lipids and domains in the bilayer. At the same time, the miscibility transition temperature of the lipid mixture is altered. Here we compare T_m in vesicles and in supported bilayers formed by rupturing the same vesicles onto glass. We determine transition temperatures using fluorescence microscopy, and identify an increase in T_m when it is measured in identical membranes in solution and on a glass surface. We systematically alter the lipid composition of our membranes in order to observe the correlation between membrane composition and variation in T_m.     

Binding of β-Amyloid (1-42) Peptide to Negatively Charged Phospholipid Membranes in the Liquid-Ordered State: Modeling and Experimental Studies

To explore the initial stages of amyloid β peptide (Aβ42) deposition on membranes, we have studied the interaction of Aβ42 in the monomeric form with lipid monolayers and with bilayers in either the liquid-disordered or the liquid-ordered (L_o) state, containing negatively charged phospholipids. Molecular dynamics (MD) simulations of the system have been performed, as well as experimental measurements. For bilayers in the L_o state, in the absence of the negatively charged lipids, interaction is weak and it cannot be detected by isothermal calorimetry. However, in the presence of phosphatidic acid, or of cardiolipin, interaction is detected by different methods and in all cases interaction is strongest with lower (2.5–5 mol %) than higher (10–20 mol %) proportions of negatively charged phospholipids. Liquid-disordered bilayers consistently allowed a higher Aβ42 binding than L_o ones. Thioflavin T assays and infrared spectroscopy confirmed a higher proportion of β-sheet formation under conditions when higher peptide binding was measured. The experimental results were supported by MD simulations. We used 100 ns MD to examine interactions between Aβ42 and three different 512 lipid bilayers consisting of palmitoylsphingomyelin, dimyristoyl phosphatidic acid, and cholesterol in three different proportions. MD pictures are different for the low- and high-charge bilayers, in the former case the peptide is bound through many contact points to the bilayer, whereas for the bilayer containing 20 mol % anionic phospholipid only a small fragment of the peptide appears to be bound. The MD results indicate that the binding and fibril formation on the membrane surface depends on the composition of the bilayer, and is the result of a subtle balance of many inter- and intramolecular interactions between the Aβ42 and membrane.     

Lipid and Peptide Dynamics in Membranes upon Insertion of n-alkyl-β-D-Glucopyranosides

The effect of nonionic detergents of the n-alkyl-β-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with ²H- and ³¹P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A_71-98, were synthesized with Ala-d₃ in the central region of the α-helix. n-Alkyl-β-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d₃ deuterated WALP-19 and GlycA_71-97 were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved ²H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD₃ methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.     

Lipid lateral diffusion and membrane heterogeneity

The pulsed field gradient (pfg)-NMR method for measurements of translational diffusion of molecules in macroscopically aligned lipid bilayers is described. This technique is proposed to have an appreciable potential for investigations in the field of lipid and membrane biology. Transport of molecules in the plane of the bilayer can be successfully studied, as well as lateral phase separation of lipids and their dynamics within the bilayer organizations. Lateral diffusion coefficients depend on lipid packing and acyl chain ordering and investigations of order parameters of perdeuterated acyl chains, using ²H NMR quadrupole splittings, are useful complements. In this review we summarize some of our recent achievements obtained on lipid membranes. In particular, bilayers exhibiting two-phase coexistence of liquid disordered (l_d) and liquid ordered (l_o) phases are considered in detail. Methods for obtaining good oriented lipid bilayers, necessary for the pfg-NMR method to be efficiently used, are also briefly described. Among our major results, besides determinations of l_d and l_o phases, belongs the finding that the lateral diffusion is the same for all components, independent of the molecular structure (including cholesterol (CHOL)), if they reside in the same domain or phase in the membrane. Furthermore, quite unexpectedly CHOL seems to partition into the l_dand l_o phases to roughly the same extent, indicating that CHOL has no strong preference for any of these phases, i.e. CHOL seems to have similar interactions with all of the lipids. We propose that the lateral phase separation in bilayers containing one high-T_m and one low-T_m lipid together with CHOL is driven by the increasing difficulty of incorporating an unsaturated or prenyl lipid into the highly ordered bilayer formed by a saturated lipid and CHOL, i.e. the phase transition is entropy driven to keep the disorder of the hydrocarbon chains of the unsaturated lipid.     

Role of cholesterol flip-flop in oxidized lipid bilayers

We performed a series of molecular dynamics simulations of cholesterol (Chol) in nonoxidized 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC) bilayer and in binary mixtures of PLPC-oxidized-lipid-bilayers with 0–50% Chol concentration and oxidized lipids with hydroperoxide and aldehyde oxidized functional groups. From the 60 unbiased molecular dynamics simulations (total of 161 μs), we found that Chol inhibited pore formation in the aldehyde-containing oxidized lipid bilayers at concentrations greater than 11%. For both pure PLPC bilayer and bilayers with hydroperoxide lipids, no pores were observed at any Chol concentration. Furthermore, increasing cholesterol concentration led to a change of phase state from the liquid-disordered to the liquid-ordered phase. This condensing effect of Chol was observed in all systems. Data analysis shows that the addition of Chol results in an increase in bilayer thickness. Interestingly, we observed Chol flip-flop only in the aldehyde-containing lipid bilayer but neither in the PLPC nor the hydroperoxide bilayers. Umbrella-sampling simulations were performed to calculate the translocation free energies and the Chol flip-flop rates. The results show that Chol’s flip-flop rate depends on the lipid bilayer type, and the highest rate are found in aldehyde bilayers. As the main finding, we shown that Chol stabilizes the oxidized lipid bilayer by confining the distribution of the oxidized functional groups.     

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.     

Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins can both decrease and increase the affinity of sterols for the lipid bilayers surrounding proteins. This is likely to affect the sterol distribution within the bilayer and thereby the lateral organization in biomembranes.     

Preparation and comparison of model bilayer systems from chloroplasts thylakoid membrane lipids for 13C-NMR studies

Model bilayer systems from individual purified chloroplast thylakoid membrane lipids, from reconstituted mixtures of these purified lipids, and from leaf total polar lipid extracts have been prepared in water, and the longitudinal relaxation times (T's₁) of the individual carbon atoms of the fatty acyl chains measured by ¹³C-NMR spectroscopy. The T's₁ increasing distance of the carbon atoms from the polar headgroups in all cases, and as the results from each of the preparations are similar, all can be used as models of chloroplast membrane bilayers. Relaxation time measurements on intact chloroplast thylakoid membranes indicate the presence of chlorophyll resonances in the ¹³C-NMR spectrum of the membrane.     

Properties of unsaturated phospholipid bilayers: Effect of cholesterol

Properties of hydrated unsaturated phosphatidylcholine (PC) lipid bilayers containing 40 mol % cholesterol and of pure PC bilayers have been studied. Various methods were applied, including molecular dynamics simulations, self-consistent field calculations, and the pulsed field gradient nuclear magnetic resonance technique. Lipid bilayers were composed of 18:0/18:1(n-9)cis PC, 18:0/18:2(n-6)cis PC, 18:0/18:3(n-3)cis PC, 18:0/20:4(n-6)cis PC, and 18:0/22:6(n-3)cis PC molecules. Lateral self-diffusion coefficients of the lipids in all these bilayers, mass density distributions of atoms and atom groups with respect to the bilayer normal, the C-H and C-C bond order parameter profiles of each phospholipid hydrocarbon chain with respect to the bilayer normal were calculated. It was shown that the lateral self-diffusion coefficient of PC molecules of the lipid bilayer containing 40 mol % cholesterol is smaller than that for a corresponding pure PC bilayer; the diffusion coefficients increase with increasing the degree of unsaturation of one of the PC chains in bilayers of both types (i.e., in pure bilayers or in bilayers with cholesterol). The presence of cholesterol in a bilayer promoted the extension of saturated and polyunsaturated lipid chains. The condensing effect of cholesterol on the order parameters was more pronounced for the double C=C bonds of polyunsaturated chains than for single C-C bonds of saturated chains.     

HIV gp41 Fusion Peptide Increases Membrane Ordering in a Cholesterol-Dependent Fashion

Fusion between viral envelopes and host cell membranes, which is mediated by special glycoproteins anchored on the viral membrane, is required for HIV viral entry and infection. The HIV gp41 fusion peptide (FP), which initiates membrane fusion, adopts either an α-helical or β-sheeted structure depending on the cholesterol concentration. We used phosphocholine spin labels on the lipid headgroup and different positions on the acyl chain to detect its perturbation on lipid bilayers containing different cholesterol concentrations by electron-spin resonance. Our findings were as follows. 1), gp41 FP affects the lipid order in the same manner as previously shown for influenza hemagglutinin FP, i.e., it has a cooperative effect versus the peptide/lipid ratio, supporting our hypothesis that membrane ordering is a common prerequisite for viral membrane fusion. 2), gp41 FP induces membrane ordering in all lipid compositions studied, whereas a nonfusion mutant FP perturbs lipid order to a significantly smaller extent. 3), In high-cholesterol-containing lipid bilayers, where gp41 FP is in the β-aggregation conformation, its effect on the lipid ordering reaches deeper into the bilayer. The different extent to which the two conformers perturb is correlated with their fusogenicity. The possible role of the two conformers in membrane fusion is discussed.     

Nanosized bilayer disks: Attractive model membranes for drug partition studies

Stable nanosized bilayer disks were prepared from either 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, or lipid mixtures with a composition reflecting that of the porcine brush border membrane. Two different polyethylene glycol (PEG)-grafted lipids, the negatively charged 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-5000] (DSPE-PEG₅₀₀₀) and the neutral N-palmitoyl-sphingosine-1-[succinyl (methoxy (polyethylene glycol) 5000] (Ceramide-PEG₅₀₀₀), were used to stabilize the disks. The disks were employed as model membranes in drug partition studies based on a fast chromatography method. Results show that the lipid composition, as well as the choice of PEG-lipid, have an important influence on the partition behavior of charged drugs. Comparative studies using multilamellar liposomes indicate that bilayer disks have the potential to generate more accurate partition data than do liposomes. Further, initial investigations using bacteriorhodopsin suggest that membrane proteins can be reconstituted into the bilayer disks. This fact further strengthens the potential of the bilayer disk as an attractive model membrane.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

Spectroscopic signatures of bilayer ordering in native biological membranes

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (L_o) and liquid-disordered (L_d) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, L_o microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In ¹³C-¹³C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.     

The N-terminal fragment of human islet amyloid polypeptide is non-fibrillogenic in the presence of membranes and does not cause leakage of bilayers of physiologically relevant lipid composition

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus (DM2). The formation of hIAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of DM2. Previous studies have shown that the N-terminal part of hIAPP, hIAPP_1-19, plays a major role in the initial interaction of hIAPP with lipid membranes. However, the exact role of this N-terminal part of hIAPP in causing membrane damage is unknown. Here we investigate the structure and aggregation properties of hIAPP_1-19 in relation to membrane damage in vitro by using membranes of the zwitterionic lipid phosphatidylcholine (PC), the anionic lipid phosphatidylserine (PS) and mixtures of these lipids to mimic membranes of islet cells. Our data reveal that hIAPP_1-19 is weakly fibrillogenic in solution and not fibrillogenic in the presence of membranes, where it adopts a secondary structure that is dependent on lipid composition and stable in time. Furthermore, hIAPP_1-19 is not able to induce leakage in membranes of PC/PS or PC bilayers, indicating that the membrane interaction of the N-terminal fragment by itself is not responsible for membrane leakage under physiologically relevant conditions. In bilayers of the anionic lipid PS, the peptide does induce membrane damage, but this leakage is not correlated to fibril formation, as it is for mature hIAPP. Hence, membrane permeabilization by the N-terminal fragment of hIAPP in anionic lipids is most likely an aspecific process, occurring via a mechanism that is not relevant for hIAPP-induced membrane damage in vivo.