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1.
Effects of mono- and di-valent metal cations on the morphology of lipid vesicles.
Hong, J, Yang, H, Pang, D, Wei, L, Deng, C
Chemistry and physics of lipids. 2018;:19-28
Abstract
Lipid vesicles are an attractive model membrane experimental platform that is widely used in a biological context. The stability of vesicles can affect their performance and depends on various experimental conditions. How bio-related ions affect vesicle morphology is poorly understood in some cases. Herein, we investigated changes in vesicle morphology influenced by cation in the static and flowing environments. The effects of different mono- and di-valent metal cations on the morphology of lipid vesicles were systematically studied using the various techniques. The results showed that divalent cations caused significant aggregation or fusion of lipid vesicles, but monovalent cations had little effect on the vesicle morphology. Cation binding increased the net surface potential of vesicles, leading to changes in the zeta potential. The same qualitative kinetics were observed for cations that had the same valence at the same ionic strength. However, different types of cations gave different quantitative effects. The order of the ability to destroy the vesicle morphology was Cu2+ > Mg2+ > Ca2+ > Na+ > K+. These results are of practical value in the use of lipid vesicles as a bionic model, and help to shed light on the role of ions at membrane surfaces and interfaces.
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2.
A simple supported tubulated bilayer system for evaluating protein-mediated membrane remodeling.
Schenk, NA, Dahl, PJ, Hanna, MG, Audhya, A, Tall, GG, Knight, JD, Anantharam, A
Chemistry and physics of lipids. 2018;:18-28
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Abstract
Fusion and fission of cellular membranes involve dramatic, protein-mediated changes in membrane curvature. Many of the experimental methods useful for investigating curvature sensing or generation require specialized equipment. We have developed a system based on supported lipid bilayers (SLBs) in which lipid tubules are simple to produce and several types of membrane remodeling events can be readily imaged using widely available instrumentation (e.g., tubule fission and/or membrane budding). Briefly, high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the SLB. After sequentially washing with water and physiological ionic strength buffer solutions, lipid tubules form spontaneously. We find that tubule formation results from solution-dependent spreading of the SLB; washing from water into physiological ionic strength buffer solution leads to expansion of the bilayer and formation of tubules. Conversely, washing from physiological buffer into water results in contraction of the membrane and loss of tubules. We demonstrate the utility of these supported tubulated bilayers, termed "STuBs," with an investigation of Sar1B, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1B to STuBs results in dramatic changes in tubule topology and eventual tubule fission. Overall, STuBs are a simple experimental system, useful for monitoring protein-mediated effects on membrane topology in real time, under physiologically relevant conditions.
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Impact of the ceramide subspecies on the nanostructure of stratum corneum lipids using neutron scattering and molecular dynamics simulations. Part I: impact of CER[NS].
Schmitt, T, Gupta, R, Lange, S, Sonnenberger, S, Dobner, B, Hauß, T, Rai, B, Neubert, RHH
Chemistry and physics of lipids. 2018;:58-68
Abstract
For this study mixtures based on the ceramides [NS] (NS = non-hydroxy-sphingosine) and [AP] (AP = α-hydroxy-phytosphingosine) in a 2:1 and 1:2 ratio, together with cholesterol and lignoceric acid, were investigated. These mixtures are modelling the uppermost skin layer, the stratum corneum. Neutron diffraction, utilizing specifically deuterated ceramide molecules, was used to obtain a maximum amount of experimental detail. Highly detailed molecular dynamics simulations were used to generate even more information from the experimental data. It was possible to observe a single lamellar phase for both systems. They had a lamellar repeat distance of 5.43 ± 0.05 nm for the [NS]/[AP] 2:1 and a slightly shorter one of 5.34 ± 0.05 nm for the 1:2 system. The structure and water content was uninfluenced by excess humidity. Both the experimental and simulation data indicated slightly tilted ceramides, with their C24 chains overlapping in the lamellar mid-plane. This arrangement is well comparable to systems investigated before. The structure of both systems, except for the differing repeat distance, looks similar at first. However, on a smaller scale there were various distinct differences, demonstrating only low redundancy between the different ceramide species, despite only minor chemical differences. The mainly ceramide [AP] determined 1:2 system has a slightly smaller repeat distance. This is a result of a tighter arrangement of the lipids chain along the bilayer normal and increased overlapping of the long chains in the lamellar middle. For the CER[NS] some novel features could be shown, despite it being the overall most investigated ceramide. These include the low adaptability to changed lateral interactions, leading to an increased chain opening. This effect could explain its low miscibility with other lipids. The investigated model systems allows it to directly compare results from the literature which have used ceramide [NS] to the most recent studies using the phytosphingosine ceramides such as ceramide [AP].
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4.
Dynamic Fingering in Adhered Lipid Membranes.
Shindell, O, Mica, N, Cheng, KH, Wang, E, Gordon, VD
Langmuir : the ACS journal of surfaces and colloids. 2018;(15):4673-4680
Abstract
Artificial lipid membranes incorporating proteins have frequently been used as models for the dynamic organization of biological structures in living cells as well as in the development of biology-inspired technologies. We report here on the experimental demonstration and characterization of a pattern-forming process that occurs in a lipid bilayer membrane adhered via biotin-avidin binding to a second lipid membrane that is supported by a solid substrate. Adhesion regions are roughly circular with a diameter of about 25 μm. Using confocal fluorescence microscopy, we record time series of dynamic fingering patterns that grow in the upper lipid membrane and intermembrane biotin-avidin bonds. The fingers are micrometer-scale elongated pores that grow from the edge of an already-stabilized hole. Finger growth is saltatory on the scale of tens of seconds. We find that as the fingers grow and the density of adhesion proteins increases, the rate of finger growth decreases exponentially and the width of newly formed fingers decreases linearly. We show that these findings are consistent with a thermodynamic description of dynamic pore formation and stabilization.
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5.
Differential targeting of membrane lipid domains by caffeic acid and its ester derivatives.
Filipe, HAL, Sousa, C, Marquês, JT, Vila-Viçosa, D, de Granada-Flor, A, Viana, AS, Santos, MSCS, Machuqueiro, M, de Almeida, RFM
Free radical biology & medicine. 2018;:232-245
Abstract
Phenolic acids have been associated to a wide range of important health benefits underlain by a common molecular mechanism of action. Considering that significant membrane permeation is prevented by their hydrophilic character, we hypothesize that their main effects result from the interplay with cell membrane surface. This hypothesis was tested using the paradigmatic caffeic acid (CA) and two of its ester derivatives, rosmarinic (RA) and chlorogenic (CGA) acids, for which we predict, based on molecular dynamics simulations, a shallow location in phospholipid bilayers dependent on the protonation-state. Using complementary experimental approaches, an interaction with the membrane was definitely revealed for the three compounds, with RA exhibiting the highest lipid bilayer partition, and the redox signals of membrane-bound RA and CA being clearly detected. Cholesterol decreased the compounds bilayer partition, but not their ability to lower membrane dipole potential. In more complex membrane models containing also sphingomyelin, with liquid disordered (ld)/ liquid ordered (lo) phases coexistence, mimicking domains in the external leaflet of human plasma membrane, all compounds were able to affect nanodomains lateral organization. RA, and to a lesser extent CGA, decreased the size of lo domains. The most significant effect of CA was the possible formation of a rigid gel-like phase, enriched in sphingomyelin. In addition, all phenolic acids decreased the order of lo domains. In sum, phenolic acid effects on the membrane are enhanced in cholesterol-rich lo phases, which predominate in the outer leaflet of human cell membranes and are involved in many key cellular processes.
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6.
Single-channel recordings of RyR1 at microsecond resolution in CMOS-suspended membranes.
Hartel, AJW, Ong, P, Schroeder, I, Giese, MH, Shekar, S, Clarke, OB, Zalk, R, Marks, AR, Hendrickson, WA, Shepard, KL
Proceedings of the National Academy of Sciences of the United States of America. 2018;(8):E1789-E1798
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Abstract
Single-channel recordings are widely used to explore functional properties of ion channels. Typically, such recordings are performed at bandwidths of less than 10 kHz because of signal-to-noise considerations, limiting the temporal resolution available for studying fast gating dynamics to greater than 100 µs. Here we present experimental methods that directly integrate suspended lipid bilayers with high-bandwidth, low-noise transimpedance amplifiers based on complementary metal-oxide-semiconductor (CMOS) integrated circuits (IC) technology to achieve bandwidths in excess of 500 kHz and microsecond temporal resolution. We use this CMOS-integrated bilayer system to study the type 1 ryanodine receptor (RyR1), a Ca2+-activated intracellular Ca2+-release channel located on the sarcoplasmic reticulum. We are able to distinguish multiple closed states not evident with lower bandwidth recordings, suggesting the presence of an additional Ca2+ binding site, distinct from the site responsible for activation. An extended beta distribution analysis of our high-bandwidth data can be used to infer closed state flicker events as fast as 35 ns. These events are in the range of single-file ion translocations.
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Does α-Tocopherol Flip-Flop Help to Protect Membranes Against Oxidation?
Boonnoy, P, Karttunen, M, Wong-Ekkabut, J
The journal of physical chemistry. B. 2018;(45):10362-10370
Abstract
α-Tocopherols (α-toc) are crucial in protecting biological membranes against oxidation by free radicals. We investigate the behavior of α-toc molecules in lipid bilayers containing oxidized lipids by molecular dynamics (MD) simulations. To verify the approach, the location and orientation of α-toc are first shown to be in agreement with previous experimental results. The simulations further show that α-toc molecules stay inside the lipid bilayer with their hydroxyl groups in contact with the bilayer surface. Interestingly, interbilayer α-toc flip-flop was observed in both oxidized and nonoxidized bilayers with significantly higher frequency in aldehyde lipid bilayer. Free-energy calculations were performed, and estimates of the flip-flop rates across the bilayers were determined. As the main finding, our results show that the presence of oxidized lipids leads to a significant decrease of free-energy barriers and that the flip-flop rates depend on the type of oxidized lipid present. Our results suggest that α-toc molecules could potentially act as high-efficacy scavengers of free radicals to protect membranes from oxidative attack and help stabilize them under oxidative stress.
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Molecular details of dimerization kinetics reveal negligible populations of transient µ-opioid receptor homodimers at physiological concentrations.
Meral, D, Provasi, D, Prada-Gracia, D, Möller, J, Marino, K, Lohse, MJ, Filizola, M
Scientific reports. 2018;(1):7705
Abstract
Various experimental and computational techniques have been employed over the past decade to provide structural and thermodynamic insights into G Protein-Coupled Receptor (GPCR) dimerization. Here, we use multiple microsecond-long, coarse-grained, biased and unbiased molecular dynamics simulations (a total of ~4 milliseconds) combined with multi-ensemble Markov state models to elucidate the kinetics of homodimerization of a prototypic GPCR, the µ-opioid receptor (MOR), embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol lipid bilayer. Analysis of these computations identifies kinetically distinct macrostates comprising several different short-lived dimeric configurations of either inactive or activated MOR. Calculated kinetic rates and fractions of dimers at different MOR concentrations suggest a negligible population of MOR homodimers at physiological concentrations, which is supported by acceptor photobleaching fluorescence resonance energy transfer (FRET) experiments. This study provides a rigorous, quantitative explanation for some conflicting experimental data on GPCR oligomerization.
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9.
Gibbs Free-Energy Gradient along the Path of Glucose Transport through Human Glucose Transporter 3.
Liang, H, Bourdon, AK, Chen, LY, Phelix, CF, Perry, G
ACS chemical neuroscience. 2018;(11):2815-2823
Abstract
Fourteen glucose transporters (GLUTs) play essential roles in human physiology by facilitating glucose diffusion across the cell membrane. Due to its central role in the energy metabolism of the central nervous system, GLUT3 has been thoroughly investigated. However, the Gibbs free-energy gradient (what drives the facilitated diffusion of glucose) has not been mapped out along the transport path. Some fundamental questions remain. Here we present a molecular dynamics study of GLUT3 embedded in a lipid bilayer to quantify the free-energy profile along the entire transport path of attracting a β-d-glucose from the interstitium to the inside of GLUT3 and, from there, releasing it to the cytoplasm by Arrhenius thermal activation. From the free-energy profile, we elucidate the unique Michaelis-Menten characteristics of GLUT3, low KM and high VMAX, specifically suitable for neurons' high and constant demand of energy from their low-glucose environments. We compute GLUT3's binding free energy for β-d-glucose to be -4.6 kcal/mol in agreement with the experimental value of -4.4 kcal/mol ( KM = 1.4 mM). We also compute the hydration energy of β-d-glucose, -18.0 kcal/mol vs the experimental data, -17.8 kcal/mol. In this, we establish a dynamics-based connection from GLUT3's crystal structure to its cellular thermodynamics with quantitative accuracy. We predict equal Arrhenius barriers for glucose uptake and efflux through GLUT3 to be tested in future experiments.
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Molecular Mechanism of Resveratrol's Lipid Membrane Protection.
Fei, Q, Kent, D, Botello-Smith, WM, Nur, F, Nur, S, Alsamarah, A, Chatterjee, P, Lambros, M, Luo, Y
Scientific reports. 2018;(1):1587
Abstract
Resveratrol, a natural compound found in red wine and various vegetables, has drawn increasing interest due to its reported benefit in cardiovascular protection, neurodegenerative disorders, and cancer therapy. The mechanism by which resveratrol exerts such pleiotropic effects remains unclear. It remains as one of the most discussed polyphenol compounds in the debating "French Paradox". In this study, using molecular dynamics simulations of dipalmitoyl phosphatidylcholine (DPPC) bilayer with resveratrol, we generated a free energy map of resveratrol's location and orientation of inside the lipid bilayer. We found that resveratrol increases the surface area per lipid and decreases membrane thickness, which is the opposite effect of the well-studied cholesterol on liquid phase DPPC. Most importantly, based on the simulation observation that resveratrol has a high probability of forming hydrogen bonds with sn-1 and sn-2 ester groups, we discovered a new mechanism using experimental approach, in which resveratrol protects both sn-1 and sn-2 ester bonds of DPPC and distearoyl phosphatidylcholine (DSPC) from phospholipase A1 (PLA1) and phospholipase A2 (PLA2) cleavage. Our study elucidates the new molecular mechanism of potential health benefits of resveratrol and possibly other similar polyphenols and provides a new paradigm for drug design based on resveratrol and its analogs.