Deuterated lipids at the ILL
Neutron scattering techniques are ideally suited for the study of lipid bilayers that are major components of cellular membranes. At the ILL, we have been working for several years to provide our users with well-characterized membrane models for physical and biological studies. While phospholipid deuteration helps elucidate membrane structure, dynamics and function, by providing selective visualisation in neutron scattering, such studies involving deuterated biomimetic membranes are currently limited by the low availability of several biologically relevant unsaturated phospholipid species.
Back in 2013, work pioneered at the ILL within the PSCM, and in collaboration with the D-Lab and Hanna Wacklin now at ESS had started and evolved over the years with the aim of extracting and purifying of PLs from deuterated cell cultures involving the following steps: (i) selection of suitable organisms for growth; (ii) Optimization of extraction protocols for PLs; (iii) Development of methods for phospholipid separation; (iv) Development of protocols for characterization of the prepared phospholipids; (v) “Mass production” for the neutron facilities user community. Funds from NMI3 and SINE2020-II are gratefully acknowledged.
So far, we have been successful in investigating methods to characterize and quantify deuterated phospholipids after producing them biologically in the oleaginous yeast, P. pastoris and the bacterium E. coli grown on perdeuterated medium.
Neutron characterization of membrane mimics from these lipids is carried out at the ILL within the LSS and SMSS groups including SANS from nanodiscs, diffraction from stacked bilayers, reflectometry from mono- and bilayers as well as within the spectroscopy group involved in inelastic scattering and spin-echo measurements from multilamellar vesicles.
Scientist Krishna Batchu has brought unique expertise on-site and since his arrival there has been enormous progress in this activity. The platform is handling already several internal requests for deuterated phospholipidss and has been heavily involved in the preparation of samples for COVID19-related science. Finally, it is worth mentioning that this activity is part of the activities currently funded by LENS. Participants to this collaborative effort include scientists Giovanna Fragneto, Krishna Batchu, Anne Martel, students Giacomo Corucci and Moritz Frewein, chemistry lab responsible Martina Sandroni. Deuterated biomass has been produced so far mainly at the ILL D-Lab by technician Valérie Laux under the supervision of Michael Haertlein.
Contact us at:
Below you will find the catalogue of lipids produced and modalities of application.
You can apply for the products listed above in aliquots of 10mg by filling an application using the template below, any time of the year, and sending it to lipids@. ill.fr
Priorities will be given to requests related to experiments to be carried out at the ILL. In this case the application will be reviewed by internal and external to the ILL scientists and in case of positive outcome the material will be provided free of charge. Co-authorship of staff at the ILL involved in the preparation of lipids will be requested.
1. Diffraction studies on natural and model lipid bilayers
F, Sebastiani; R, Harvey; S, Khanniche; J.B, Artero; M, Haertlein and Fragneto, G
The European Physical Journal Special Topics. 12/2012; 213:355-365
In this study we have used neutron diffraction to examine the swelling behaviour and bilayer parameters of membranes reconstituted from polar lipids extracted from B. subtilis and model systems composed of synthetic phospholipids. Evidence for phase separation in the model system (lacking in Lysyl-PG, L-PG) is discussed in relation to its possible contribution to membrane domain formation through lipid-lipid interactions. Comparing these results with those obtained from the bilayers composed of lipids extracted from bacterial cells gives us some indication of the role of L-PG in the B. subtilis plasma membrane.
2. Production and Analysis of Perdeuterated Lipids from Pichia pastoris Cells
de Ghellinck, A; Schaller, H; Laux, V; Haertlein, M; Sferrazza, M; Wacklin, H; Johuet, J and Fragneto, G
Plos One Volume: 9 Issue: 4 Article Number: e92999 Published: APR 18 2014
Probing molecules using perdeuteration (i.e deuteration in which all hydrogen atoms are replaced by deuterium) is extremely useful in a wide range of biophysical techniques. In the case of lipids, the synthesis of the biologically relevant unsaturated perdeuterated lipids is challenging and not usually pursued. In this work, perdeuterated phospholipids and sterols from the yeast Pichia pastoris grown in deuterated medium are extracted and analyzed as derivatives by gas chromatography and mass spectrometry respectively. When yeast cells are grown in a deuterated environment, the phospholipid homeostasis is maintained but the fatty acid unsaturation level is modified while the ergosterol synthesis is not affected by the deuterated culture medium. Our results confirm that the production of well defined natural unsaturated perdeuterated lipids is possible and gives also new insights about the process of desaturase enzymes.
3. Multi-lamellar organization of fully deuterated lipid extracts of yeast membranes
Gerelli, Y; de Ghellinck, Alexis; Jouhet, J; Laux, V; Haertlein, M and Fragneto, G
Acta Crystallographica Section D Biological Crystallography12/2014; 70(12):3167.
Neutron scattering studies on mimetic biomembranes are currently limited by the low availability of deuterated unsaturated lipid species. In the present work, results from the first neutron diffraction experiments on fully deuterated lipid extracts from the yeast Pichia pastoris are presented. The structural features of these fully deuterated lipid stacks are compared with those of their hydrogenous analogues and with other similar synthetic systems. The influence of temperature and humidity on the samples has been investigated by means of small momentum-transfer neutron diffraction. All of the lipid extracts investigated self-assemble into multi-lamellar stacks having different structural periodicities; the stacking distances are affected by temperature and humidity without altering the basic underlying arrangement. At high relative humidity the deuterated and hydrogenous samples are similar in their multi-lamellar arrangement, being characterized by two main periodicities of ∼75 and ∼110 Å reflecting the presence of a large number of polar phospholipid molecules. Larger differences are found at lower relative humidity, where hydrogenous lipids are characterized by a larger single lamellar structure than that observed in the deuterated samples. In both cases the heterogeneity in composition is reflected in a wide structural complexity. The different behaviour upon dehydration can be related to compositional differences in the molecular composition of the two samples, which is attributed to metabolic effects related to the use of perdeuterated growth media.
Keywords: full deuteration; lipid extracts; neutron scattering.
4. Lipid polyunsaturation determines the extent of membrane structural changes induced by Amphotericin B in Pichia pastoris yeast
de Ghellinck, A; Fragneto, G; Laux, V; Haertlein, M; Jouhet, J; Sferrazza, M and Wacklin, H
Biochimica et Biophysica Acta 06/2015; DOI:10.1016/j.bbamem.2015.06.006
The activity of the potent but highly toxic antifungal drug Amphotericin B (AmB), used intravenously to treat systemic fungal and parasitic infections, is widely accepted to result from its specific interaction with the fungal sterol ergosterol. While the effect of sterols on AmB activity has been intensely investigated, the role of membrane phospholipid composition has largely been ignored, and structural studies of native membranes have been hampered by their complex and disordered nature. We show for the first time that the structure of fungal membranes derived from Pichia pastoris yeast depends on the degree of lipid polyunsaturation, which has an impact on the structural consequences of AmB activity. AmB inserts in yeast membranes even in the absence of ergosterol, and forms an extra-membraneous layer whose thickness is resolved to be 4-5 nm. In ergosterol-containing membranes, AmB insertion is accompanied by ergosterol extraction into this layer. The AmB-sponge mediated depletion of ergosterol from P. pastoris membranes gives rise to a significant membrane thinning effect that depends on the degree of lipid polyunsaturation. The resulting hydrophobic mismatch is likely to interfere with a much broader range of membrane protein functions than those directly involving ergosterol, and suggests that polyunsaturated lipids could boost the efficiency of AmB. Furthermore, a low degree of lipid polyunsaturation leads to least AmB insertion and may protect host cells against the toxic effects of AmB. These results provide a new framework based on lipid composition and membrane structure through which we can understand its antifungal action and develop better treatments.
Keywords: Amphotericin B; Neutron reflectometry; Pichia pastoris; Yeast lipids.
5. The influence of mild acidity on lysyl-phosphatidylglycerol biosynthesis and lipid membrane physico-chemical properties in methicillin-resistant Staphylococcus aureus
Rehal Reg, P; Marbach, H; Hubbard, A; Sacranie, Anam A; Sebastiani, F; Fragneto, G & Harvey, R.D.
Chemistry and Physics of Lipids 2017, 206, 60-70
The increased biosynthesis of lysyl-phosphatidylglycerol in Staphylococcus aureus when cultured under conditions of mild acidity and the resultant increased proportion of this lipid in the plasma membrane of the bacterium, alters the physico-chemical properties of lipid bilayers in a manner which is itself dependent upon environmental pH. Clinically relevant strains of S. aureus, both methicillin susceptible and resistant, all exhibited increased lysyl-phosphatidylglycerol biosynthesis in response to mild environmental acidity, albeit to differing degrees, from ∼30% to ∼55% total phospholipid. Polar lipid extracts from these bacteria were analysed by 31P NMR and reconstituted into vesicles and monolayers, which were characterised by zeta potential measurements and Langmuir isotherms respectively. A combination of increased lysyl-phosphatidylglycerol content and mild environmental acidity were found to synergistically neutralise the charge of the membranes, in one instance altering the zeta potential from -56mV to +21mV, and induce closer packing between the lipids. Challenge of reconstituted S. aureus lipid model membranes by the antimicrobial peptide magainin 2 F5W was examined using monolayer subphase injection and neutron diffraction, and revealed that ionisation of the headgroup α-amine of lysyl-phosphatidylglycerol at pH 5.5, which reduced the magnitude of the peptide-lipid interaction by 80%, was more important for resisting peptide partitioning than increased lipid content alone. The significance of these results is discussed in relation to how colonising mildly acidic environments such as human mucosa may be facilitated by increased lysyl-phosphatidylglycerol biosynthesis and the implications of this for further biophysical analysis of the role of this lipid in bacterial membranes.
Keywords: Antimicrobial resistance; Lysyl-phosphatidylglycerol; Mild acidity; Monolayers; Neutron diffraction.
6. The impact of deuteration on natural and synthetic lipids: A neutron diffraction study
Luchini, A; Delhom, R; Deme, B and Fragneto, G
Colloids and Surfaces B-Biointerfaces 2018, 168: 126-133
The structural investigation of cellular membranes requires access to model systems where the molecular complexity is representative of the cellular environment and that allow for the exploitation of structural techniques. Neutron scattering, and in particular neutron diffraction can provide unique and detailed information on the structure of lipid membranes. However, deuterated samples are desirable to fully exploit this powerful method. Recently, the extraction of lipids from microorganisms grown in deuterated media was demonstrated to be both an attracting route to obtain complex lipid mixtures resembling the composition of natural membranes, and to producing deuterated molecules in a very convenient way. A full characterization of these deuterated extracts is hence pivotal for their use in building up model membrane systems. Here we report the structural characterization of lipid extracts obtained from Pichia pastoris by means of neutron diffraction measurements. In particular, we compare the structure of membranes extracted from yeast cells grown in a standard culture medium and in a corresponding deuterated culture medium. The results show that the different molecular composition of the deuterated and protiated lipid extracts induce different structural organization of the lipid membranes. In addition, we compare these membranes composed of extracted yeast lipids with stacked bilayers prepared from synthetic lipid mixtures.
Keywords: Cholesterol; Deuteration; Ergosterol; Neutron diffraction; Yeast lipids.
7. Neutrons and model membranes: Moving towards complexity
Fragneto, G; Delhom, R; Joly, L and Scoppola, E
Current Opinion in Colloid & Interface Science 2018, 38:108–121
Colloids and Surfaces B-Biointerfaces 2018, 168: 126-133
Cells, the basic units of living organisms, are well delineated and separated from the external environment by membranes. Capable of both enclosing the cellular constituents and allowing exchanges with the outside world, these membranes are only a few nanometers thick. All the membranes in a human body cover an area of a few hectares, but account for only a small part of our mass. To study the dynamics and function of these amazing objects, physicists first seek to understand their structure. This involves experiments on model systems, simpler and better controlled than real membranes, and can profit from a probe that is able to access different scales of size and time: thermal neutrons. Since the pioneering work in the seventies on cell membrane structure by neutron scattering, developments driven by constantly improving neutron instrumentation, coupled with development of measurement and analysis methods, have involved both the optimization of samples towards more biologically relevant model systems and include the use of more complex lipid mixtures up to natural extracts. This review does not have the ambition to address the large number of contributions from all the groups working in this area in research laboratories and neutron facilities. It gives an update on some studies in the field carried out mainly by the authors and collaborators.
8. Biocompatible Glyconanoparticles by Grafting of Sophorolipid Monolayers on Monodisperse Iron Oxide Nanoparticles.
Lassenberger, A; Batchu, KC and Reimhult, E
ACS Appl. Bio Mater. 2019, 2, 7, 3095–3107
This work presents the synthesis and characterization of sophorolipid-coated monodisperse iron oxide nanoparticles. Sophorolipids are biological glycosylated amphiphiles produced by the yeast S. bombicola. In their open acidic form, sophorolipids have been used as a surface stabilizing agent for metal and metal oxide nanoparticles but with a poor control over size and structural properties. In this work, the COOH function of sophorolipids (SL) was modified with nitrodopamine (NDA), a catechol known for its high affinity to iron ions. The resulting new form of sophorolipid–nitrodopamide (SL-NDA) was used as a surface ligand for monodisperse iron oxide nanoparticles. We show by a combination of thermogravimetric analysis and small-angle X-ray and neutron scattering that iron oxide nanoparticles (IONP) are stabilized by a single, high-density SL-NDA layer. This results in excellent colloidal stability under biologically relevant conditions, such as at high protein and salt concentrations. The IONP grafted with SL-NDA showed a negligible uptake by cells and no cytotoxicity, which was tested on two representative cell lines. Thus, they reveal the potential of sophorolipids as stable and nontoxic surface coatings for IONP-based biomedical and biotechnological applications.
9. Creating Asymmetric Phospholipid Vesicles via Exchange with Lipid-Coated Silica Nanoparticles
Liu, Y; Kelley, EG; Batchu, KC; Porcar, L and Salas-Perez, U
Langmuir2020, 36, 30, 8865–8873
Recently, effort has been placed into fabricating model free-floating asymmetric lipid membranes, such as asymmetric vesicles. Here, we report on the use of lipid-coated silica nanoparticles to exchange lipids with initially symmetric vesicles to generate composition-controlled asymmetric vesicles. Our method relies on the simple and natural exchange of lipids between membranes through an aqueous medium. Using a selected temperature, time, and ratio of lipid-coated silica nanoparticles to vesicles, we produced a desired highly asymmetric leaflet composition. At this point, the silica nanoparticles were removed by centrifugation, leaving the asymmetric vesicles in solution. In the present work, the asymmetric vesicles were composed of isotopically distinct dipalmitoylphosphatidylcholine lipids. Lipid asymmetry was detected by both small-angle neutron scattering (SANS) and proton nuclear magnetic resonance (1H NMR). The rate at which the membrane homogenizes at 75 °C was also assessed.
10. The Antifungal Mechanism of Amphotericin B Elucidated in Ergosterol and Cholesterol-Containing Membranes Using Neutron Reflectometry
Delhom, R; Nelson, A; Laux, V; [...] Wacklin-Knecht, AP
Nanomaterials, December 2020
We have characterized and compared the structures of ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes before and after interaction with the amphiphilic antifungal drug amphotericin B (AmB) using neutron reflection. AmB inserts into both pure POPC and sterol-containing membranes in the lipid chain region and does not significantly perturb the structure of pure POPC membranes. By selective per-deuteration of the lipids/sterols, we show that AmB extracts ergosterol but not cholesterol from the bilayers and inserts to a much higher degree in the cholesterol-containing membranes. Ergosterol extraction by AmB is accompanied by membrane thinning. Our results provide new insights into the mechanism and antifungal effect of AmB in these simple models of fungal and mammalian membranes and help understand the molecular origin of its selectivity and toxic side effects.
Keywords: POPC; amphotericin B; cholesterol; ergosterol; lipid membranes; neutron reflection.
11. Peptide Discs as Precursors of Biologically Relevant Supported Lipid Bilayers
Luchini, A; Sebastiani, F; Tidemand, F; Batchu, KC; Fragneto, G and Arleth, L
Journal of Colloid and Interface Science, Volume 585, March 2021, Pages 376-385
Supported lipid bilayers (SLBs) are commonly used to investigate the structure and dynamics of biological membranes. Vesicle fusion is a widely exploited method to produce SLBs. However, this process becomes less favoured when the vesicles contain complex lipid mixtures, e.g. natural lipid extracts. In these cases, it is often necessary to change experimental parameters, such as temperature, to unphysiological values to trigger the SLB formation. This may induce lipid degradation and is also not compatible with including membrane proteins or other biomolecules into the bilayers. Here, we show that the peptide discs, ~10 nm discoidal lipid bilayers stabilized in solution by a self-assembled 18A peptide belt, can be used as precursors for SLBs. The characterizations by means of neutron reflectometry and attenuated total reflectance-FTIR spectroscopy show that SLBs were successfully formed both from synthetic lipid mixtures (surface coverage 90-95%) and from natural lipid mixtures (surface coverage ~85%). Traces of 18A peptide (below 0.02 M ratio) left at the support surface after the bilayer formation do not affect the SLB structure. Altogether, we demonstrate that peptide disc formation of SLBs is much faster than the SLB formation by vesicle fusion and without the need of altering any experimental variable from physiologically relevant values.
Keywords: ATR-FTIR; Neutron reflectometry; Peptide discs; Supported lipid bilayers.
12. Structural Characterization of Natural Yeast Phosphatidylcholine and Bacterial Phosphatidylglycerol Lipid Multilayers by Neutron Diffraction
Luchini, A; Corucci, G; Batchu, KC; Laux, V; Haertlein, M, Crisitiglio V and Fragneto, G
Frontiers in Chemistry 2021, 9, 85
Eukaryotic and prokaryotic cell membranes are difficult to characterize directly with biophysical methods. Membrane model systems, that include fewer molecular species, are therefore often used to reproduce their fundamental chemical and physical properties. In this context, natural lipid mixtures directly extracted from cells are a valuable resource to produce advanced models of biological membranes for biophysical investigations and for the development of drug testing platforms. In this study we focused on single phospholipid classes, i.e. Pichia pastoris phosphatidylcholine (PC) and Escherichia coli phosphatidylglycerol (PG) lipids. These lipids were characterized by a different distribution of their respective acyl chain lengths and number of unsaturations. We produced both hydrogenous and deuterated lipid mixtures. Neutron diffraction experiments at different relative humidities were performed to characterize multilayers from these lipids and investigate the impact of the acyl chain composition on the structural organization. The novelty of this work resides in the use of natural extracts with a single class head-group and a mixture of chain compositions coming from yeast or bacterial cells. The characterization of the PC and PG multilayers showed that, as a consequence of the heterogeneity of their acyl chain composition, different lamellar phases are formed.
13. Lipid bilayer degradation induced by SARS-CoV-2 spike protein as revealed by neutron reflectometry
Luchini, A; Micciulla, S; Corucci, G; Batchu, KC; Santamaria, A; Laux, V; Darwish, T; Russell, RA; Thepaut, M; Bally, I; Fieschi, F and Fragneto, G
Scientific Reports, volume 11, Article number: 14867 (2021)
SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
14. Strikingly different roles of SARS-CoV-2 fusion peptides uncovered by neutron scattering.
Santamaria, A; Batchu, KC; Matsarskaia, O; Prévost, SF; Russo, D; Natali, F; Seydel, T; Hoffmann, I; Laux, V; Haertlein, M; Darwish, TA; Russell, RA; Corucci, G; Fragneto, G; Maestro, A; and Zaccai, NR.
J Am Chem Soc. Volume 144 (7), Feb 2022, Pages 2968-2979)
Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin-echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium however re-orients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.
15. Interdigitation-induced Order and Disorder in Asymmetric Membranes.
Frewein, MPK; Piller, P; Semeraro, EF; Batchu, KC; Heberle, FA; Scott, H; Gerelli, Y; Porcar, L; and Pabst, G.
Journal of Membrane Biology, 2022) [doi: 10.1007/s00232-022-00234-0]
We studied the transleaflet coupling of compositionally asymmetric liposomes in the fluid phase. The vesicles were produced by cyclodextrin-mediated lipid exchange and contained dipalmitoyl phosphatidylcholine (DPPC) in the inner leaflet and different mixed-chain phosphatidylcholines (PCs) as well as milk sphingomyelin (MSM) in the outer leaflet. In order to jointly analyse the obtained small-angle neutron and X-ray scattering data, we adapted existing models of trans-bilayer structures to measure the overlap of the hydrocarbon chain termini by exploiting the contrast of the terminal methyl ends in X-ray scattering. In all studied systems, the bilayer-asymmetry has large effects on the lipid packing density. Fully saturated mixed-chain PCs interdigitate into the DPPC-containing leaflet and evoke disorder in one or both leaflets. The long saturated acyl chains of MSM penetrate even deeper into the opposing leaflet, which in turn has an ordering effect on the whole bilayer. These results are qualitatively understood in terms of a balance of entropic repulsion of fluctuating hydrocarbon chain termini and van der Waals forces, which is modulated by the interdigitation depth. Monounsaturated PCs in the outer leaflet also induce disorder in DPPC despite vestigial or even absent interdigitation. Instead, the transleaflet coupling appears to emerge here from a matching of the inner leaflet lipids to the larger lateral lipid area of the outer leaflet lipids.
16. Investigation on the relationship between lipid composition and out-of-plane structure in model membranes composed of extracted natural phospholipids.
Santamaria, A; Batchu, KC; Laux, V; Haertlein, M; Darwish, T; Fragneto, G; Zaccai, N; Maestro,A.
(Submitted to Chemical Sciences, 2022).
Unravelling the structural diversity of cellular membranes is a paramount challenge in life sciences. In particular, lipid composition affects the membrane’s collective behaviour, as well as its interactions with other biological molecules. In this work, the relationship between membrane composition and resultant structural features was investigated by surface-pressure area isotherms and neutron reflectometry on in vitro membrane models of the mammalian Plasma and endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) membranes in the form of Langmuir monolayers. Natural extracted yeast lipids were used as, unlike synthetic lipids, the acyl chain saturation pattern of yeast and mammalian lipids are similar. The results demonstrated that the out-of-plane structure of the model membranes, as well as their lateral packing, depend strongly on their specific composition, with cholesterol having a major influence on the in-plane morphology, yielding a coexistence of liquid-order and liquid-disorder phases.
17. Slow flip-flop of cholesterol in membranes and implications for its distribution across the plasma membrane.
Liu, Y; Pergande, M; Batchu, KC, Kelley, EG; Cologna, S; Porcar, L and Salas-Perez, U.
(Submitted to Biophysical Journal, 2022)
Trans-bilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, distinctive, actively maintained, and functionally important, the trans-bilayer distribution of PM cholesterol is not reliably established. Using a model membrane consisting of the common cell membrane lipid palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), we were able to produce vesicles with an asymmetric cholesterol composition in each leaflet. The resulting asymmetry and redistribution of cholesterol in the bilayer through trans-bilayer flip-flop has been characterized and followed using solution 1H NMR. We found that homogenization of cholesterol across the membrane took many hours at physiological temperatures. This finding suggests that sidedness of cholesterol distribution in the PM is energetically feasible.
18. Developing advanced models of biological membranes withhydrogenous and deuterated natural glycerophospholipids extracted from yeast.
(Manuscript to be submitted to Chemistry and Physics of Lipids).
Batchu, KC; Corucci, G; Luchini, A; Santamaria, A; Frewein, M; Laux, V; Haertlein, M; Botte, C; Yamaryo-Botte, Y; Tully, M; Maestro, A; Martel, A; Porcar, L; and Fragneto, G.