Publications by authors named "Frederick A Heberle"

53 Publications

Dataset of asymmetric giant unilamellar vesicles prepared via hemifusion: Observation of anti-alignment of domains and modulated phases in asymmetric bilayers.

Data Brief 2021 Apr 3;35:106927. Epub 2021 Mar 3.

Cornell University, United States.

The data provided with this paper are confocal fluorescence images of symmetric giant unilamellar vesicles (GUVs) and asymmetric giant unilamellar vesicles (aGUVs). In this work, aGUVs were prepared using the hemifusion method and are labelled with two different fluorescent dyes, named TFPC and DiD. Both dyes show strong preference for the liquid-disordered (Ld) phase instead of the liquid-ordered (Lo) phase. The partition of these dyes favoring the Ld phase leads to bright Ld phase and dark Lo phase domains in symmetric GUVs observed by fluorescence microscopy. In symmetric vesicles, the bright and the dark domains of the inner and the outer leaflets are aligned. In aGUVs, the fluorescent probe TFPC exclusively labels the aGUV outer leaflet. Here, we show a dataset of fluorescence micrographs obtained using scanning fluorescence confocal microscopy. For the system chosen, the fluorescence signal of TFPC and DiD show anti-alignment of the brighter domains on aGUVs. Important for this dataset, TFPC and DiD have fluorescence emission centered in the green and far-red region of the visible spectra, respectively, and the dyes' fluorescence emission bands do not overlap. This dataset were collected in the same conditions of the dataset reported in the co-submitted work (Enoki, et al. 2021) where most of aGUVs show domains alignment. In addition, we show micrographs of GUVs displaying modulated phases and macrodomains. We also compare the modulated phases observed in GUVs and aGUVs. For these datasets, we collected a sequence of micrographs using confocal microscopy varying the -position, termed a -stack. Images were collected in a scanning microscope Nikon Eclipse C2+ (Nikon Instruments, Melville, NY). Additional samples used to measure the lipid concentrations and to prepare GUVs with accurate lipid fractions are also provided with this paper.
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http://dx.doi.org/10.1016/j.dib.2021.106927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973298PMC
April 2021

Investigation of the domain line tension in asymmetric vesicles prepared via hemifusion.

Biochim Biophys Acta Biomembr 2021 Jun 26;1863(6):183586. Epub 2021 Feb 26.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, United States of America.

The plasma membrane (PM) is asymmetric in lipid composition. The distinct and characteristic lipid compositions of the exoplasmic and cytoplasmic leaflets lead to different lipid-lipid interactions and physical-chemical properties in each leaflet. The exoplasmic leaflet possesses an intrinsic ability to form coexisting ordered and disordered fluid domains, whereas the cytoplasmic leaflet seems to form a single fluid phase. To better understand the interleaflet interactions that influence domains, we compared asymmetric model membranes that capture salient properties of the PM with simpler symmetric membranes. Using asymmetric giant unilamellar vesicles (aGUVs) prepared by hemifusion with a supported lipid bilayer, we investigate the domain line tension that characterizes the behavior of coexisting ordered + disordered domains. The line tension can be related to the contact perimeter of the different phases. Compared to macroscopic phase separation, the appearance of modulated phases was found to be a robust indicator of a decrease in domain line tension. Symmetric GUVs of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC)/cholesterol (chol) were formed into aGUVs by replacing the GUV outer leaflet with DOPC/chol = 0.8/0.2 in order to create a cytoplasmic leaflet model. These aGUVs revealed lower line tension for the ordered + disordered domains of the exoplasmic model leaflet.
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http://dx.doi.org/10.1016/j.bbamem.2021.183586DOI Listing
June 2021

FRET from phase-separated vesicles: An analytical solution for a spherical geometry.

Chem Phys Lipids 2020 11 13;233:104982. Epub 2020 Oct 13.

Department of Chemistry, University of Tennessee, Knoxville, TN 37996, United States. Electronic address:

Förster resonance energy transfer (FRET) is a powerful tool for investigating heterogeneity in lipid bilayers. In model membrane studies, samples are frequently unilamellar vesicles with diameters of 20-200 nm. It is well-known that FRET efficiency is insensitive to vesicle curvature in uniformly mixed lipid bilayers, and consequently theoretical models for FRET typically assume a planar geometry. Here, we use a spherical harmonic expansion of the acceptor surface density to derive an analytical solution for FRET between donor and acceptor molecules distributed on the surface of a sphere. We find excellent agreement between FRET predicted from the model and FRET calculated from corresponding Monte Carlo simulations, thus validating the model. An extension of the model to the case of a non-uniform acceptor surface density (i.e., a phase-separated vesicle) reveals that FRET efficiency depends on vesicle size when acceptors partition between the coexisting phases, and approaches the efficiency of a uniformly mixed bilayer as the vesicle size decreases. We show that this is an indirect effect of constrained domain size, rather than an intrinsic effect of vesicle curvature. Surprisingly, the theoretical predictions were not borne out in experiments: we did not observe a statistically significant change in FRET efficiency in phase-separated vesicles as a function of vesicle size. We discuss factors that likely mask the vesicle size effect in extruded samples.
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http://dx.doi.org/10.1016/j.chemphyslip.2020.104982DOI Listing
November 2020

How cholesterol stiffens unsaturated lipid membranes.

Proc Natl Acad Sci U S A 2020 09 25;117(36):21896-21905. Epub 2020 Aug 25.

Department of Physics, Virginia Tech, Blacksburg, VA 24061;

Cholesterol is an integral component of eukaryotic cell membranes and a key molecule in controlling membrane fluidity, organization, and other physicochemical parameters. It also plays a regulatory function in antibiotic drug resistance and the immune response of cells against viruses, by stabilizing the membrane against structural damage. While it is well understood that, structurally, cholesterol exhibits a densification effect on fluid lipid membranes, its effects on membrane bending rigidity are assumed to be nonuniversal; i.e., cholesterol stiffens saturated lipid membranes, but has no stiffening effect on membranes populated by unsaturated lipids, such as 1,2-dioleoyl--glycero-3-phosphocholine (DOPC). This observation presents a clear challenge to structure-property relationships and to our understanding of cholesterol-mediated biological functions. Here, using a comprehensive approach-combining neutron spin-echo (NSE) spectroscopy, solid-state deuterium NMR (H NMR) spectroscopy, and molecular dynamics (MD) simulations-we report that cholesterol locally increases the bending rigidity of DOPC membranes, similar to saturated membranes, by increasing the bilayer's packing density. All three techniques, inherently sensitive to mesoscale bending fluctuations, show up to a threefold increase in effective bending rigidity with increasing cholesterol content approaching a mole fraction of 50%. Our observations are in good agreement with the known effects of cholesterol on the area-compressibility modulus and membrane structure, reaffirming membrane structure-property relationships. The current findings point to a scale-dependent manifestation of membrane properties, highlighting the need to reassess cholesterol's role in controlling membrane bending rigidity over mesoscopic length and time scales of important biological functions, such as viral budding and lipid-protein interactions.
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http://dx.doi.org/10.1073/pnas.2004807117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486787PMC
September 2020

Direct label-free imaging of nanodomains in biomimetic and biological membranes by cryogenic electron microscopy.

Proc Natl Acad Sci U S A 2020 08 5;117(33):19943-19952. Epub 2020 Aug 5.

Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, TX 77030;

The nanoscale organization of biological membranes into structurally and compositionally distinct lateral domains is believed to be central to membrane function. The nature of this organization has remained elusive due to a lack of methods to directly probe nanoscopic membrane features. We show here that cryogenic electron microscopy (cryo-EM) can be used to directly image coexisting nanoscopic domains in synthetic and bioderived membranes without extrinsic probes. Analyzing a series of single-component liposomes composed of synthetic lipids of varying chain lengths, we demonstrate that cryo-EM can distinguish bilayer thickness differences as small as 0.5 Å, comparable to the resolution of small-angle scattering methods. Simulated images from computational models reveal that features in cryo-EM images result from a complex interplay between the atomic distribution normal to the plane of the bilayer and imaging parameters. Simulations of phase-separated bilayers were used to predict two sources of contrast between coexisting ordered and disordered phases within a single liposome, namely differences in membrane thickness and molecular density. We observe both sources of contrast in biomimetic membranes composed of saturated lipids, unsaturated lipids, and cholesterol. When extended to isolated mammalian plasma membranes, cryo-EM reveals similar nanoscale lateral heterogeneities. The methods reported here for direct, probe-free imaging of nanodomains in unperturbed membranes open new avenues for investigation of nanoscopic membrane organization.
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http://dx.doi.org/10.1073/pnas.2002200117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443941PMC
August 2020

Laterally Resolved Small-Angle Scattering Intensity from Lipid Bilayer Simulations: An Exact and a Limited-Range Treatment.

J Chem Theory Comput 2020 Aug 14;16(8):5287-5300. Epub 2020 Jul 14.

Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland 20892, United States.

When combined, molecular simulations and small-angle scattering experiments are able to provide molecular-scale resolution of structure. Separately, scattering experiments provide only intermingled pair correlations between atoms, while molecular simulations are limited by model quality and the relatively short time scales that they can access. Their combined strength relies on agreement between the experimental spectra and those computed by simulation. To date, computing the neutron spectra from a molecular simulation of a lipid bilayer is straightforward only if the structure is approximated by laterally averaging the in-plane bilayer structure. However, this neglects all information about lateral heterogeneity, e.g., clustering of components in a lipid mixture. This paper presents two methods for computing the scattering intensity of simulated bilayers with in-plane heterogeneity, enabling a full treatment of both the transverse and lateral bilayer structure for the first time. The first method, termed the Dirac Brush, computes the exact spectra including spurious artifacts resulting from using information from neighboring periodic cells to account for the long-range structure of the bilayer. The second method, termed PFFT, applies a mean-field treatment in the field far from a scattering element, resulting in a correlation range that can be tuned (eliminating correlations with neighboring periodic images), but with computational cost that prohibits obtaining the exact (Dirac Brush) spectra. Following their derivation, the two methods are applied to a coarse-grained molecular simulation of a bilayer inhomogeneity, demonstrating the contributions of lateral correlations to the resulting spectra.
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http://dx.doi.org/10.1021/acs.jctc.0c00132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587404PMC
August 2020

Molecular Structure of Sphingomyelin in Fluid Phase Bilayers Determined by the Joint Analysis of Small-Angle Neutron and X-ray Scattering Data.

J Phys Chem B 2020 06 16;124(25):5186-5200. Epub 2020 Jun 16.

Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996, United States.

We have determined the fluid bilayer structure of palmitoyl sphingomyelin (PSM) and stearoyl sphingomyelin (SSM) by simultaneously analyzing small-angle neutron and X-ray scattering data. Using a newly developed scattering density profile (SDP) model for sphingomyelin lipids, we report structural parameters including the area per lipid, total bilayer thickness, and hydrocarbon thickness, in addition to lipid volumes determined by densitometry. Unconstrained all-atom simulations of PSM bilayers at 55 °C using the C36 CHARMM force field produced a lipid area of 56 Å, a value that is 10% lower than the one determined experimentally by SDP analysis (61.9 Å). Furthermore, scattering form factors calculated from the unconstrained simulations were in poor agreement with experimental form factors, even though segmental order parameter () profiles calculated from the simulations were in relatively good agreement with profiles obtained from NMR experiments. Conversely, constrained area simulations at 61.9 Å resulted in good agreement between the simulation and experimental scattering form factors, but not with profiles from NMR. We discuss possible reasons for the discrepancies between these two types of data that are frequently used as validation metrics for molecular dynamics force fields.
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http://dx.doi.org/10.1021/acs.jpcb.0c03389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7458099PMC
June 2020

Lipid Rafts: Controversies Resolved, Mysteries Remain.

Trends Cell Biol 2020 05 20;30(5):341-353. Epub 2020 Feb 20.

Department of Chemistry, University of Tennessee, Knoxville, TN 37996, USA; Shull Wollan Center, Oak Ridge National Laboratory, Oak Ridge, TN 33830, USA.

The lipid raft hypothesis postulates that lipid-lipid interactions can laterally organize biological membranes into domains of distinct structures, compositions, and functions. This proposal has in equal measure exhilarated and frustrated membrane research for decades. While the physicochemical principles underlying lipid-driven domains has been explored and is well understood, the existence and relevance of such domains in cells remains elusive, despite decades of research. Here, we review the conceptual underpinnings of the raft hypothesis and critically discuss the supporting and contradicting evidence in cells, focusing on why controversies about the composition, properties, and even the very existence of lipid rafts remain unresolved. Finally, we highlight several recent breakthroughs that may resolve existing controversies and suggest general approaches for moving beyond questions of the existence of rafts and towards understanding their physiological significance.
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http://dx.doi.org/10.1016/j.tcb.2020.01.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7798360PMC
May 2020

The structures of polyunsaturated lipid bilayers by joint refinement of neutron and X-ray scattering data.

Chem Phys Lipids 2020 07 12;229:104892. Epub 2020 Feb 12.

Department of Physics, Brock University, St. Catharines, ON, Canada; The Bredesen Center, University of Tennessee, Knoxville, TN, USA; Shull Wollan Center, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Large Scale Structures Group, Neutron Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Department of Physics and Astronomy, University of Tennessee, Knoxville, TN, USA. Electronic address:

We present the detailed structural analysis of polyunsaturated fatty acid-containing phospholipids namely, 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PDPC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (SDPC). A newly developed molecular dynamics (MD) simulation parsing scheme for lipids containing fatty acids with multiple double bonds was implemented into the scattering density profile (SDP) model to simultaneously refine differently contrasted neutron and X-ray scattering data. SDP analyses of scattering data at 30 °C yielded lipid areas of 71.1 Å and 70.4 Å for PDPC and SDPC bilayers, respectively, and a model free analysis of PDPC at 30 °C resulted in a lipid area of 72 Å. In addition to bilayer structural parameters, using area-constrained MD simulations we determined the area compressibility modulus, K, to be 246.4 mN/m, a value similar to other neutral phospholipids.
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http://dx.doi.org/10.1016/j.chemphyslip.2020.104892DOI Listing
July 2020

The antioxidant vitamin E as a membrane raft modulator: Tocopherols do not abolish lipid domains.

Biochim Biophys Acta Biomembr 2020 08 15;1862(8):183189. Epub 2020 Jan 15.

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario,Canada; Department of Physics, University of Windsor, Windsor, Ontario, Canada. Electronic address:

The antioxidant vitamin E is a commonly used vitamin supplement. Although the multi-billion dollar vitamin and nutritional supplement industry encourages the use of vitamin E, there is very little evidence supporting its actual health benefits. Moreover, vitamin E is now marketed as a lipid raft destabilizing anti-cancer agent, in addition to its antioxidant behaviour. Here, we studied the influence of vitamin E and some of its vitamers on membrane raft stability using phase separating unilamellar lipid vesicles in conjunction with small-angle scattering techniques and fluorescence microscopy. We find that lipid phase behaviour remains unperturbed well beyond physiological concentrations of vitamin E (up to a mole fraction of 0.10). Our results are consistent with a proposed line active role of vitamin E at the domain boundary. We discuss the implications of these findings as they pertain to lipid raft modification in native membranes, and propose a new hypothesis for the antioxidant mechanism of vitamin E.
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http://dx.doi.org/10.1016/j.bbamem.2020.183189DOI Listing
August 2020

Transverse lipid organization dictates bending fluctuations in model plasma membranes.

Nanoscale 2020 Jan;12(3):1438-1447

Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.

Membrane undulations play a vital role in many biological processes, including the regulation of membrane protein activity. The asymmetric lipid composition of most biological membranes complicates theoretical description of these bending fluctuations, yet experimental data that would inform any such a theory is scarce. Here, we used neutron spin-echo (NSE) spectroscopy to measure the bending fluctuations of large unilamellar vesicles (LUV) having an asymmetric transbilayer distribution of high- and low-melting lipids. The asymmetric vesicles were prepared using cyclodextrin-mediated lipid exchange, and were composed of an outer leaflet enriched in egg sphingomyelin (ESM) and an inner leaflet enriched in 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE), which have main transition temperatures of 37 °C and 25 °C, respectively. The overall membrane bending rigidity was measured at three temperatures: 15 °C, where both lipids are in a gel state; 45 °C, where both lipids are in a fluid state; and 30 °C, where there is gel-fluid co-existence. Remarkably, the dynamics for the fluid asymmetric LUVs (aLUVs) at 30 °C and 45 °C do not follow trends predicted by their symmetric counterparts. At 30 °C, compositional asymmetry suppressed the bending fluctuations, with the asymmetric bilayer exhibiting a larger bending modulus than that of symmetric bilayers corresponding to either the outer or inner leaflet. We conclude that the compositional asymmetry and leaflet coupling influence the internal dissipation within the bilayer and result in membrane properties that cannot be directly predicted from corresponding symmetric bilayers.
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http://dx.doi.org/10.1039/c9nr07977gDOI Listing
January 2020

With Lipid Rafts, Context Is Everything.

Biophys J 2019 11 25;117(9):1549-1551. Epub 2019 Sep 25.

Department of Chemistry, University of Tennessee, Knoxville, Tennessee. Electronic address:

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http://dx.doi.org/10.1016/j.bpj.2019.07.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838737PMC
November 2019

On the Mechanism of Bilayer Separation by Extrusion, or Why Your LUVs Are Not Really Unilamellar.

Biophys J 2019 10 16;117(8):1381-1386. Epub 2019 Sep 16.

Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee; Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas. Electronic address:

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Submicroscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would impact interpretations from many model membrane experiments. Using three probe-free methods-small angle X-ray and neutron scattering and cryogenic electron microscopy-we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100-nm diameter pores. In such preparations, only ∼35% of lipids are externally accessible and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein the formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.
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http://dx.doi.org/10.1016/j.bpj.2019.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817544PMC
October 2019

Peptide-Induced Lipid Flip-Flop in Asymmetric Liposomes Measured by Small Angle Neutron Scattering.

Langmuir 2019 09 27;35(36):11735-11744. Epub 2019 Aug 27.

Department of Chemistry and Biochemistry , University of Windsor , Windsor , N9B 3P4 ON Canada.

Despite the prevalence of lipid transbilayer asymmetry in natural plasma membranes, most biomimetic model membranes studied are symmetric. Recent advances have helped to overcome the difficulties in preparing asymmetric liposomes in vitro, allowing for the examination of a larger set of relevant biophysical questions. Here, we investigate the stability of asymmetric bilayers by measuring lipid flip-flop with time-resolved small-angle neutron scattering (SANS). Asymmetric large unilamellar vesicles with inner bilayer leaflets containing predominantly 1-palmitoyl-2-oleoyl--glycero-3-phosphocholine (POPC) and outer leaflets composed mainly of 1,2-dimyristoyl--glycero-3-phosphocholine (DMPC) displayed slow spontaneous flip-flop at 37 C (half-time, = 140 h). However, inclusion of peptides, namely, gramicidin, alamethicin, melittin, or pHLIP (i.e., pH-low insertion peptide), accelerated lipid flip-flop. For three of these peptides (i.e., pHLIP, alamethicin, and melittin), each of which was added externally to preformed asymmetric vesicles, we observed a completely scrambled bilayer in less than 2 h. Gramicidin, on the other hand, was preincorporated during the formation of the asymmetric liposomes and showed a time resolvable 8-fold increase in the rate of lipid asymmetry loss. These results point to a membrane surface-related (e.g., adsorption/insertion) event as the primary driver of lipid scrambling in the asymmetric model membranes of this study. We discuss the implications of membrane peptide binding, conformation, and insertion on lipid asymmetry.
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http://dx.doi.org/10.1021/acs.langmuir.9b01625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393738PMC
September 2019

Phosphatidylserine Asymmetry Promotes the Membrane Insertion of a Transmembrane Helix.

Biophys J 2019 04 19;116(8):1495-1506. Epub 2019 Mar 19.

Department of Biochemistry & Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee. Electronic address:

The plasma membrane (PM) contains an asymmetric distribution of lipids between the inner and outer bilayer leaflets. A lipid of special interest in eukaryotic membranes is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM, but PS redistributes to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, the influence of PS asymmetry on membrane protein structure and folding are poorly understood. The pH low insertion peptide (pHLIP) adsorbs to the membrane surface at a neutral pH, but it inserts into the membrane at an acidic pH. We have previously observed that in symmetric vesicles, PS affects the membrane insertion of pHLIP by lowering the pH midpoint of insertion. Here, we studied the effect of PS asymmetry on the membrane interaction of pHLIP. We developed a modified protocol to create asymmetric vesicles containing PS and employed Annexin V labeled with an Alexa Fluor 568 fluorophore as a new probe to quantify PS asymmetry. We observed that the membrane insertion of pHLIP was promoted by the asymmetric distribution of negatively charged PS, which causes a surface charge difference between bilayer leaflets. Our results indicate that lipid asymmetry can modulate the formation of an α-helix on the membrane. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important aspects of protein-membrane interactions.
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http://dx.doi.org/10.1016/j.bpj.2019.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486527PMC
April 2019

Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid Vesicles.

Biophys J 2019 03 25;116(5):860-873. Epub 2019 Jan 25.

Department of Physiology and Biophysics, Weill Cornell Medical College, New York, New York.

Unlike most transmembrane proteins, phospholipids can migrate from one leaflet of the membrane to the other. Because this spontaneous lipid translocation (flip-flop) tends to be very slow, cells facilitate the process with enzymes that catalyze the transmembrane movement and thereby regulate the transbilayer lipid distribution. Nonenzymatic membrane-spanning proteins with unrelated primary functions have also been found to accelerate lipid flip-flop in a nonspecific manner and by various hypothesized mechanisms. Using deuterated phospholipids, we examined the acceleration of flip-flop by gramicidin channels, which have well-defined structures and known functions, features that make them ideal candidates for probing the protein-membrane interactions underlying lipid flip-flop. To study compositionally and isotopically asymmetric proteoliposomes containing gramicidin, we expanded a recently developed protocol for the preparation and characterization of lipid-only asymmetric vesicles. Channel incorporation, conformation, and function were examined with small angle x-ray scattering, circular dichroism, and a stopped-flow spectrofluorometric assay, respectively. As a measure of lipid scrambling, we used differential scanning calorimetry to monitor the effect of gramicidin on the melting transition temperatures of the two bilayer leaflets. The two calorimetric peaks of the individual leaflets merged into a single peak over time, suggestive of scrambling, and the effect of the channel on the transbilayer lipid distribution in both symmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and asymmetric 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles was quantified from proton NMR measurements. Our results show that gramicidin increases lipid flip-flop in a complex, concentration-dependent manner. To determine the molecular mechanism of the process, we used molecular dynamics simulations and further computational analysis of the trajectories to estimate the extent of membrane deformation. Together, the experimental and computational approaches were found to constitute an effective means for studying the effects of transmembrane proteins on lipid distribution in both symmetric and asymmetric model membranes.
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http://dx.doi.org/10.1016/j.bpj.2019.01.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400823PMC
March 2019

Preparation of asymmetric phospholipid vesicles for use as cell membrane models.

Nat Protoc 2018 09;13(9):2086-2101

Department of Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada.

Freely suspended liposomes are widely used as model membranes for studying lipid-lipid and protein-lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet's lipid composition: the overall lipid composition is determined by gas chromatography-mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.
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http://dx.doi.org/10.1038/s41596-018-0033-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459367PMC
September 2018

Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation.

Biochim Biophys Acta Biomembr 2018 10 3;1860(10):1985-1993. Epub 2018 May 3.

Department of Nutrition, Gillings School of Global Public Health and School of Medicine, The University of North Carolina at Chapel Hill, United States. Electronic address:

Docosahexaenoic acid (DHA, 22:6) is an n-3 polyunsaturated fatty acid (n-3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.
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http://dx.doi.org/10.1016/j.bbamem.2018.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218320PMC
October 2018

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases.

Biophys J 2018 04;114(8):1921-1935

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (∼200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5-10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ∼5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.
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http://dx.doi.org/10.1016/j.bpj.2018.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937166PMC
April 2018

Intrinsic Curvature-Mediated Transbilayer Coupling in Asymmetric Lipid Vesicles.

Biophys J 2018 01;114(1):146-157

University of Graz, Institute of Molecular Biosciences, Biophysics Division, NAWI Graz; BioTechMed-Graz, Graz, Austria. Electronic address:

We measured the effect of intrinsic lipid curvature, J, on structural properties of asymmetric vesicles made of palmitoyl-oleoyl-phosphatidylethanolamine (POPE; J<0) and palmitoyl-oleoyl-phosphatidylcholine (POPC; J∼0). Electron microscopy and dynamic light scattering were used to determine vesicle size and morphology, and x-ray and neutron scattering, combined with calorimetric experiments and solution NMR, yielded insights into leaflet-specific lipid packing and melting processes. Below the lipid melting temperature we observed strong interleaflet coupling in asymmetric vesicles with POPE inner bilayer leaflets and outer leaflets enriched in POPC. This lipid arrangement manifested itself by lipids melting cooperatively in both leaflets, and a rearrangement of lipid packing in both monolayers. On the other hand, no coupling was observed in vesicles with POPC inner bilayer leaflets and outer leaflets enriched in POPE. In this case, the leaflets melted independently and did not affect each other's acyl chain packing. Furthermore, we found no evidence for transbilayer structural coupling above the melting temperature of either sample preparation. Our results are consistent with the energetically preferred location of POPE residing in the inner leaflet, where it also resides in natural membranes, most likely causing the coupling of both leaflets. The loss of this coupling in the fluid bilayers is most likely the result of entropic contributions.
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http://dx.doi.org/10.1016/j.bpj.2017.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773765PMC
January 2018

Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties.

Biophys J 2017 Nov;113(9):2004-2015

Department of Biochemistry and Molecular Cell Biology, Cornell University, Ithaca, New York. Electronic address:

Binding of the retroviral structural protein Gag to the cellular plasma membrane is mediated by the protein's matrix (MA) domain. Prominent among MA-PM interactions is electrostatic attraction between the positively charged MA domain and the negatively charged plasma membrane inner leaflet. Previously, we reported that membrane association of HIV-1 Gag, as well as purified Rous sarcoma virus (RSV) MA and Gag, depends strongly on the presence of acidic lipids and is enhanced by cholesterol (Chol). The mechanism underlying this enhancement was unclear. Here, using a broad set of in vitro and in silico techniques we addressed molecular mechanisms of association between RSV MA and model membranes, and investigated how Chol enhances this association. In neutron scattering experiments with liposomes in the presence or absence of Chol, MA preferentially interacted with preexisting POPS-rich clusters formed by nonideal lipid mixing, binding peripherally to the lipid headgroups with minimal perturbation to the bilayer structure. Molecular dynamics simulations showed a stronger MA-bilayer interaction in the presence of Chol, and a large Chol-driven increase in lipid packing and membrane surface charge density. Although in vitro MA-liposome association is influenced by disparate variables, including ionic strength and concentrations of Chol and charged lipids, continuum electrostatic theory revealed an underlying dependence on membrane surface potential. Together, these results conclusively show that Chol affects RSV MA-membrane association by making the electrostatic potential at the membrane surface more negative, while decreasing the penalty for lipid headgroup desolvation. The presented approach can be applied to other viral and nonviral proteins.
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http://dx.doi.org/10.1016/j.bpj.2017.08.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5685651PMC
November 2017

Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in Synthetic and Natural Composition Lipid Membranes.

Langmuir 2017 09 5;33(38):10016-10026. Epub 2017 Sep 5.

Department of Mechanical, Aerospace, and Biomedical Engineering, and §Department of Physics and Astronomy, The University of Tennessee , Knoxville, Tennessee 37996, United States.

In-plane lipid organization and phase separation in natural membranes play key roles in regulating many cellular processes. Highly cooperative, first-order phase transitions in model membranes consisting of few lipid components are well understood and readily detectable via calorimetry, densitometry, and fluorescence. However, far less is known about natural membranes containing numerous lipid species and high concentrations of cholesterol, for which thermotropic transitions are undetectable by the above-mentioned techniques. We demonstrate that membrane capacitance is highly sensitive to low-enthalpy thermotropic transitions taking place in complex lipid membranes. Specifically, we measured the electrical capacitance as a function of temperature for droplet interface bilayer model membranes of increasing compositional complexity, namely, (a) a single lipid species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some ternary compositions, capacitance exhibited an abrupt, temperature-dependent change that coincided with the transition detected by other techniques. In addition, capacitance measurements revealed transitions in mixed-lipid membranes that were not detected by the other techniques. Most notably, capacitance measurements of bTLE bilayers indicated a transition at ∼38 °C not seen with any other method. Likewise, capacitance measurements detected transitions in some well-studied ternary mixtures that, while known to yield coexisting lipid phases, are not detected with calorimetry or densitometry. These results indicate that capacitance is exquisitely sensitive to low-enthalpy membrane transitions because of its sensitivity to changes in bilayer thickness that occur when lipids and excess solvent undergo subtle rearrangements near a phase transition. Our findings also suggest that heterogeneity confers stability to natural membranes that function near transition temperatures by preventing unwanted defects and macroscopic demixing associated with high-enthalpy transitions commonly found in simpler mixtures.
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http://dx.doi.org/10.1021/acs.langmuir.7b02022DOI Listing
September 2017

Complex biomembrane mimetics on the sub-nanometer scale.

Biophys Rev 2017 Aug 17;9(4):353-373. Epub 2017 Jul 17.

Institute of Molecular Biosciences, Biophysics Division, NAWI Graz, University of Graz, 8010, Graz, Austria.

Biomimetic lipid vesicles are indispensable tools for gaining insight into the biophysics of cell physiology on the molecular level. The level of complexity of these model systems has steadily increased, and now spans from domain-forming lipid mixtures to asymmetric lipid bilayers. Here, we review recent progress in the development and application of elastic neutron and X-ray scattering techniques for studying these systems in situ and under physiologically relevant conditions on the nanometer to sub-nanometer length scales. In particular, we focus on: (1) structural details of coexisting liquid-ordered and liquid-disordered domains, including their thickness and lipid packing mismatch as a function of a size transition from nanoscopic to macroscopic domains; (2) membrane-mediated protein partitioning into lipid domains; (3) the role of the aqueous medium in tuning interactions between membranes and domains; and (4) leaflet-specific structure in asymmetric bilayers and passive lipid flip-flop.
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http://dx.doi.org/10.1007/s12551-017-0275-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578918PMC
August 2017

Line Tension Controls Liquid-Disordered + Liquid-Ordered Domain Size Transition in Lipid Bilayers.

Biophys J 2017 Apr;112(7):1431-1443

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.
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http://dx.doi.org/10.1016/j.bpj.2017.02.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390056PMC
April 2017

Joint small-angle X-ray and neutron scattering data analysis of asymmetric lipid vesicles.

J Appl Crystallogr 2017 Apr 28;50(Pt 2):419-429. Epub 2017 Feb 28.

Instiute of Molecular Biosciences, Biophysics Division, University of Graz, Austria; BioTechMed-Graz, Graz, 8010, Austria.

Low- and high-resolution models describing the internal transbilayer structure of asymmetric lipid vesicles have been developed. These models can be used for the joint analysis of small-angle neutron and X-ray scattering data. The models describe the underlying scattering length density/electron density profiles either in terms of slabs or through the so-called scattering density profile, previously applied to symmetric lipid vesicles. Both models yield structural details of asymmetric membranes, such as the individual area per lipid, and the hydrocarbon thickness of the inner and outer bilayer leaflets. The scattering density profile model, however, comes at a cost of increased computational effort but results in greater structural resolution, showing a slightly lower packing of lipids in the outer bilayer leaflet of ∼120 nm diameter palmitoyl-oleoyl phosphatidyl-choline (POPC) vesicles, compared to the inner leaflet. Analysis of asymmetric dipalmitoyl phosphatidylcholine/POPC vesicles did not reveal evidence of transbilayer coupling between the inner and outer leaflets at 323 K, above the melting transition temperature of the two lipids.
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http://dx.doi.org/10.1107/S1600576717000656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377341PMC
April 2017

H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers.

Langmuir 2017 04 3;33(15):3731-3741. Epub 2017 Feb 3.

Institute of Molecular Biosciences, Biophysics Division, NAWI Graz, University of Graz , Graz 8010, Austria.

We measured the transbilayer diffusion of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in large unilamellar vesicles, in both the gel (L) and fluid (L) phases. The choline resonance of headgroup-protiated DPPC exchanged into the outer leaflet of headgroup-deuterated DPPC-d13 vesicles was monitored using H NMR spectroscopy, coupled with the addition of a paramagnetic shift reagent. This allowed us to distinguish between the inner and outer bilayer leaflet of DPPC, to determine the flip-flop rate as a function of temperature. Flip-flop of fluid-phase DPPC exhibited Arrhenius kinetics, from which we determined an activation energy of 122 kJ mol. In gel-phase DPPC vesicles, flip-flop was not observed over the course of 250 h. Our findings are in contrast to previous studies of solid-supported bilayers, where the reported DPPC translocation rates are at least several orders of magnitude faster than those in vesicles at corresponding temperatures. We reconcile these differences by proposing a defect-mediated acceleration of lipid translocation in supported bilayers, where long-lived, submicron-sized holes resulting from incomplete surface coverage are the sites of rapid transbilayer movement.
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http://dx.doi.org/10.1021/acs.langmuir.6b04485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397887PMC
April 2017

Lipid bilayer thickness determines cholesterol's location in model membranes.

Soft Matter 2016 Nov;12(47):9417-9428

Department of Physics, Brock University, St. Catharines, Ontario L2S 3A1, Canada and The Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee, Knoxville, Tennessee 37996, USA and Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA. and Shull Wollan Center-a Joint Institute for Neutron Sciences, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA and Department of Physics and Astronomy, University of Tennessee, Knoxville, Tennessee 37996, USA.

Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of different lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. These results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.
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http://dx.doi.org/10.1039/c6sm01777kDOI Listing
November 2016

Morphology-Induced Defects Enhance Lipid Transfer Rates.

Langmuir 2016 09 12;32(38):9757-64. Epub 2016 Sep 12.

Institute of Molecular Biosciences, Biophysics Division, NAWI Graz, University of Graz , Graz 8010, Austria.

Molecular transfer between nanoparticles has been considered to have important implications regarding nanoparticle stability. Recently, the interparticle spontaneous lipid transfer rate constant for discoidal bicelles was found to be very different from spherical, unilamellar vesicles (ULVs). Here, we investigate the mechanism responsible for this discrepancy. Analysis of the data indicates that lipid transfer is entropically favorable, but enthalpically unfavorable with an activation energy that is independent of bicelle size and long- to short-chain lipid molar ratio. Moreover, molecular dynamics simulations reveal a lower lipid dissociation energy cost in the vicinity of interfaces ("defects") induced by the segregation of the long- and short-chain lipids in bicelles; these defects are not present in ULVs. Taken together, these results suggest that the enhanced lipid transfer observed in bicelles arises from interfacial defects as a result of the hydrophobic mismatch between the long- and short-chain lipid species. Finally, the observed lipid transfer rate is found to be independent of nanoparticle stability.
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http://dx.doi.org/10.1021/acs.langmuir.6b02099DOI Listing
September 2016

Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.

Langmuir 2016 05 16;32(20):5195-200. Epub 2016 May 16.

Institute of Molecular Biosciences, Biophysics Division, NAWI Graz, University of Graz , Graz 8010, Austria.

Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer asymmetry in modulating membrane lateral organization.
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http://dx.doi.org/10.1021/acs.langmuir.5b04562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910133PMC
May 2016
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