Vesicle budding caused by lysolipid-induced asymmetry stress.

Lysolipids such as lauroyl, myristoyl, and palmitoyl lysophosphatidylcholine (LPC) insert into the outer leaflet of liposomes but do not flip to the inner leaflet over many hours. This way, they create asymmetry stress between the intrinsic areas of the two leaflets. We have studied how this stress is relaxed with particular emphasis on the budding and fission of small (diameter 20-30 nm) daughter vesicles (DVs). Asymmetric flow field-flow fractionation was utilized to quantify the extent of budding from large unilamellar vesicles after exposure to LPC. Budding starts at a low threshold of the order of 2 mol% LPC in the outer (and ≈0 mol% LPC in the inner) leaflet. We see reason to assume that the fractional fluorescence intensity from DVs is a good approximation for the fraction of membrane lipid, POPC, transferred into DVs. Accordingly, budding starts with a "budding power" of ≈6 POPC molecules budding off per LPC added, corresponding to a more than 10-fold accumulation of LPC in the outer leaflet of DVs to ≈24 mol%. As long as budding is possible, little strain is built up in the membranes, a claim supported by the lack of changes in limiting fluorescence anisotropy, rotational correlation time, and fluorescence lifetime of symmetrically and asymmetrically inserted TMA-DPH. At physiological osmolarity, budding is typically limited to 20-30% of budded fraction with some batch-to-batch variation, but independent of the LPC species. We hypothesize that the budding limit is determined by the excess area of the liposomes upon preparation, which is then used up upon budding given the larger area-to-volume ratio of smaller liposomes. As the mother vesicles approach ideal spheres, budding must stop. This is qualitatively supported by increased and decreased budding limits of osmotically predeflated and preinflated vesicles, respectively.

Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment.

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral-yet water-soluble-polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein-lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

Filling the Gap with Long n-Alkanes: Incorporation of C20 and C30 into Phospholipid Membranes.

Investigating how hydrophobic molecules mix with phospholipid bilayers and how they affect membrane properties is commonplace in biophysics. Despite this, a molecular-level empirical description of a membrane model as simple as a phospholipid bilayer with long linear hydrophobic chains incorporated is still missing. Here, we present an unprecedented molecular characterization of the incorporation of two long n-alkanes, n-eicosane (C20) and n-triacontane (C30) with 20 and 30 carbons, respectively, in phosphatidylcholine (PC) bilayers using a combination of experimental techniques (2H NMR, 31P NMR, 1H-13C dipolar recoupling solid-state NMR, X-ray scattering, and cryogenic electron microscopy) and atomistic molecular dynamics (MD) simulations. At low hydration, deuterated C20 and C30 yield 2H NMR spectra evidencing anisotropic-motion, which demonstrates their miscibility in PC membranes up to a critical alkane-to-acyl-chain volume fraction, ϕc. The acquired 2H NMR spectra of C20 and C30 have notably different lineshapes. At low alkane volume fractions below ϕc, CHARMM36 MD simulations predict such 2H NMR spectra qualitatively and thus enable an atomistic-level interpretation of the spectra. Above ϕc, the 2H NMR lineshapes become characteristic of motions in the intermediate-regime that, together with the MD simulation results, suggest the onset of immiscibility between the alkane molecules and the acyl chains. For all the systems investigated, the phospholipid molecular structure is unperturbed by the presence of the alkanes. However, at conditions of excess hydration and at surprisingly low alkane fractions below ϕc, a peak characteristic of isotropic motion is observed in both the 2H spectra of the alkanes and 31P spectra of the phospholipids, strongly indicating that the incorporation of the alkanes induces a reduction on the average radius of the lipid vesicles.

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs.

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein-protein and protein-lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenous─as opposed to overexpressed─membrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane-protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane-protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane-protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes.

Measuring protein insertion areas in lipid monolayers by fluorescence correlation spectroscopy.

Membrane insertion of protein domains is an important step in many membrane remodeling processes, for example, in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. To our knowledge, this is the first optical measurement of this quantity on a system in equilibrium with direct determination of the number of inserted protein and no further assumptions concerning the binding thermodynamics. Whereas macroscopic total area changes in lipid monolayers are typically measured on a Langmuir film balance, finding the number of inserted proteins without perturbing the system and quantitating any small area changes has posed a challenge. Here, we address both issues by performing two-color fluorescence correlation spectroscopy directly on the monolayer. With a fraction of the protein being fluorescently labeled, the number of inserted proteins is determined in situ without resorting to invasive techniques such as collecting the monolayer by aspiration. The second color channel is exploited to monitor a small fraction of labeled lipids to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2 was obtained for Sar1 in monolayers from a lipid mixture typically used in COPII reconstitution experiments, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to also be applied to laterally heterogeneous monolayers and bilayers.

Self-Assembly of Protein-Containing Lipid-Bilayer Nanodiscs from Small-Molecule Amphiphiles.

When membrane proteins are removed from their natural environment, the quality of the membrane-solubilizing agent used is critical for preserving their native structures and functions. Nanodiscs that retain a lipid-bilayer core around membrane proteins have attracted great attention because they offer a much more native-like environment than detergent micelles. Here, two small-molecule amphiphiles with diglucose headgroups and either a hydrocarbon or a fluorocarbon hydrophobic chain are shown to directly assemble lipids and membrane proteins to form native nanodiscs rather than mixed micelles. Self-assembly of nanodiscs of increasing complexity from both defined, artificial vesicles as well as complex, cellular membranes is demonstrated. A detailed investigation of bilayer integrity and membrane-protein activity in these nanodiscs reveals gentle effects on the encapsulated bilayer core. The fluorinated amphiphile appears particularly promising because its lipophobicity results in gentle, non-perturbing interactions with the nanoscale lipid bilayer. A sequential model of nanodisc self-assembly is proposed that proceeds through perforation of the original membrane followed by saturation and complete solubilization of the bilayer. On this basis, pseudophase diagrams are established for mixtures of lipids and nanodisc-forming diglucoside amphiphiles, and the latter are used for the extraction of a broad range of membrane proteins from cellular membranes.

A Diazirine-Modified Membrane Lipid to Study Peptide/Lipid Interactions - Chances and Challenges.

Although incorporation of photo-activatable lipids into membranes potentially opens up novel avenues for investigating interactions with proteins, the question of whether diazirine-modified lipids are suitable for such studies, remains under debate. Focusing on the potential for studying lipid/peptide interactions by cross-linking mass spectrometry (XL-MS), we developed a diazirine-modified lipid (DiazPC), and examined its behaviour in membranes incorporating the model α-helical peptide LAVA20. We observed an unexpected backfolding of the diazirine-containing stearoyl chain of the lipid. This surprising behaviour challenges the potential application of DiazPC for future XL-MS studies of peptide and protein/lipid interactions. The observations made for DiazPC most likely represent a general phenomenon for any type of membrane lipids with a polar moiety incorporated into the alkyl chain. Our finding is therefore of importance for future protein/lipid interaction studies relying on modified lipid probes.

Nanoscale Model System for the Human Myelin Sheath.

Neurodegenerative disorders are among the most common diseases in modern society. However, the molecular bases of diseases such as multiple sclerosis or Charcot-Marie-Tooth disease remain far from being fully understood. Research in this field is limited by the complex nature of native myelin and by difficulties in obtaining good in vitro model systems of myelin. Here, we introduce an easy-to-use model system of the myelin sheath that can be used to study myelin proteins in a native-like yet well-controlled environment. To this end, we present myelin-mimicking nanodiscs prepared through one of the amphiphilic copolymers styrene/maleic acid (SMA), diisobutylene/maleic acid (DIBMA), and styrene/maleimide sulfobetaine (SMA-SB). These nanodiscs were tested for their lipid composition using chromatographic (HPLC) and mass spectrometric (MS) methods and, utilizing spin probes within the nanodisc, their comparability with liposomes was studied. In addition, their binding behavior with bovine myelin basic protein (MBP) was scrutinized to ensure that the nanodiscs represent a suitable model system of myelin. Our results suggest that both SMA and SMA-SB are able to solubilize the myelin-like (cytoplasmic) liposomes without preferences for specific lipid headgroups or fatty acyl chains. In nanodiscs of both SMA and SMA-SB (called SMA(-SB)-lipid particles, short SMALPs or SMA-SBLPs, respectively), the polymers restrict the lipids' motion in the hydrophobic center of the bilayer. The headgroups of the lipids, however, are sterically less hindered in nanodiscs when compared with liposomes. Myelin-like SMALPs are able to bind bovine MBP, which can stack the lipid bilayers like in native myelin, showing the usability of these simple, well-controlled systems in further studies of protein-lipid interactions of native myelin.

Lipid-Dependent Interaction of Human N-BAR Domain Proteins with Sarcolemma Mono- and Bilayers.

The N-BAR domain of the human Bin1 protein is indispensable for T-tubule biogenesis in skeletal muscles. It binds to lipid mono- and bilayers that mimic the sarcolemma membrane composition, and it transforms vesicles into uniform tubules by generating a decorating protein scaffold. We found that Δ(1-33)BAR, lacking the N-terminal amphipathic helix (H0), and H0 alone bind to sarcolemma monolayers, although both proteins are not able to tubulate sarcolemma vesicles. By variation of the lipid composition, we elucidated the role of PI(4,5)P2, cholesterol, and an asymmetric sarcolemma composition for Bin1-N-BAR binding and sarcolemma tubulation. Our results indicate that Bin1-N-BAR binding is low in the absence of PI(4,5)P2 and it is affected by additional changes in the negative headgroup charge and lipid acyl chain composition. However, it is not dependent on the cholesterol content. The results from Langmuir monolayer experiments are complementary to lipid bilayer studies using electron microscopy that provides information on membrane curvature generation.

Tuning the Thickness of a Biomembrane by Stapling Diamidophospholipids with Bolalipids.

In a biological membrane, proteins require specific lipids of distinctive length and chain saturation surrounding them. The active tuning of the membrane thickness therefore opens new possibilities in the study and manipulation of membrane proteins. Here, we introduce the concept of stapling phospholipids to different degrees of interdigitation depth by mixing 1,3-diamidophospholipids with single-chain bolalipids. The mixed membranes were studied by calorimetric assays, electron microscopy, X-ray, and infrared measurements to provide a complete biophysical characterization of membrane stapling. The matching between the diamidophospholipids and the bolalipids can be so strong as to completely induce a new phase that is more stable than the gel phase of the individual components.

Synthesis and aggregation behaviour of single-chain, 1,32-alkyl-branched bis(phosphocholines) - part 2: lateral chain length triggers self-assembling from sheets to fibres to vesicles.

Six single-chain, 1,32-alkyl-branched bis(phosphocholines) PC-C32(1,32Cm)-PC have been synthesized as model lipids for naturally occurring archaeal membrane lipids. The preparation of these bipolar amphiphiles bearing lateral alkyl chains of different lengths (C4-C15) was realized using a Cu-catalyzed Grignard bis-coupling reaction of various primary alkyl-branched bromides as side parts and a 1,22-dibromide as the centre part. The aggregation behaviour of these bolalipids in water was initially investigated by differential scanning calorimetry and transmission electron microscopy. As a main result, the types of aggregates found and their stability upon heating were strongly connected to the length of the lateral alkyl chain of the bolalipid: short and long lateral chains led to lamellar structures, whereas side chains of medium length led to fibrous aggregates. In future, these bolalipids could be used to produce tailored and stabilized liposomes for oral drug delivery.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) nanodispersions as potential anti-inflammatory therapeutics: Comparison of in vitro activity and impact of pegylation.

Phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with putative anti-inflammatory potential. However, studies comparing PS and PG are rare and were mainly conducted with phospholipid-dispersions of large size and broad distributions. Thus, we prepared small-sized PS- and PG-loaded liposomes exhibiting narrow distribution, and additionally studied the impact of liposome-pegylation on the reduction of the TNFα-production caused by the PS- and PG-liposomes. These PS- and PG-containing nanodispersions had a small size around 100nm and a narrow distribution (PDI<0.1). The liposome-dispersions showed no toxicity in NHDF- and 3T3-cells and virtually no hemolytic activity. They decreased the TNFα-production of LPS-(lipopolysaccharide)-stimulated mouse peritoneal macrophages in vitro. PG-liposomes always decreased the TNFα-levels more potently than PS-liposomes. Pegylation of PS- and PG-liposomes caused different Zeta potentials, but did not change biological activity. The results of the current study indicate a high potential of the tested formulations for phospholipid-based anti-inflammatory therapies.

Integrative biology of native cell extracts: a new era for structural characterization of life processes.

Advances in electron microscopy have provided unprecedented access to the structural characterization of large, flexible and heterogeneous complexes. Until recently, cryo-electron microscopy (cryo-EM) has been applied to understand molecular organization in either highly purified, isolated biomolecules or in situ. An emerging field is developing, bridging the gap between the two approaches, and focuses on studying molecular organization in native cell extracts. This field has demonstrated its potential by resolving the structure of fungal fatty acid synthase (FAS) at 4.7 Å [Fourier shell correlation (FSC) = 0.143]; FAS was not only less than 50% enriched, but also retained higher-order binders, previously unknown. Although controversial in the sense that the lysis step might introduce artifacts, cell extracts preserve aspects of cellular function. In addition, cell extracts are accessible, besides cryo-EM, to modern proteomic methods, chemical cross-linking, network biology and biophysical modeling. We expect that automation in imaging cell extracts, along with the integration of molecular/cell biology approaches, will provide remarkable achievements in the study of closer-to-life biomolecular states of pronounced biotechnological and medical importance. Such steps will, eventually, bring us a step closer to the biophysical description of cellular processes in an integrative, holistic approach.

Azide-Modified Membrane Lipids: Miscibility with Saturated Phosphatidylcholines.

In this study, we describe the miscibility of four azide-modified membrane phospholipids (azidolipids) with conventional phospholipids. The azidolipids bear an azide group at different positions of the sn-1 or sn-2 alkyl chain and they further differ in the type of linkage (ester vs ether) of the sn-2 alkyl chain. Investigations regarding the miscibility of the azidolipids with bilayer-forming phosphatidylcholines will evaluate lipid mixtures that are suitable for the production of stable azidolipid-doped liposomes. These vesicles then serve as model membranes for the incorporation of model peptides or proteins in the future. The miscibility of both types of phospholipids was studied by calorimetric assays, electron microscopy, small-angle X-ray scattering, infrared spectroscopy, and dynamic light scattering to provide a complete biophysical characterization of the mixed systems.

Hybrid Double-Chain Maltose-Based Detergents: Synthesis and Colloidal and Biochemical Evaluation.

Four hybrid double-chain surfactants with a maltose polar head were synthesized. The apolar domain consists of a hydrogenated chain, and a partially fluorinated chain made of a propyl hydrogenated spacer terminated by a perfluorinated core of various lengths. Their water solubility was found to be lower than 1 g/L irrespective of the length of both chains. The self-assembling properties of pure hybrids in water were studied by dynamic light scattering and transmission electron microscopy, which revealed the formation of two populations of aggregates with diameters of 8-50 nm and 80-300 nm. When mixed with the classical detergent n-dodecylmaltoside (DDM), the four hybrids were well soluble and formed small mixed micelles. DDM/hybrid mixtures were further evaluated for the extraction of the full-length, wild-type human GPCR adenosine receptor (A2AR), and the bacterial transporter AcrB. The solubilization of A2AR showed extraction efficiencies ranging from 40 to 70%, while that of AcrB reached 60-90%. Finally, three of the hybrids exhibited significant thermostabilization when present as additives. The derivative with a C12-hydrogenated chain and a C4F9-fluorinated chain emerged as the most potent additive exhibiting both good extraction yields of A2AR and AcrB and thermostabilization of A2AR by ∼7 °C.

Impact of Headgroup Asymmetry and Protonation State on the Aggregation Behavior of a New Type of Glycerol Diether Bolalipid.

In the present work, we describe the synthesis and the temperature-dependent aggregation behavior of a new class of asymmetrical glycerol diether bolalipids. These bolalipids are composed of a membrane-spanning alkyl chain with 32 carbon atoms (C32) in the sn-3 position, a methyl-branched C16 alkyl chain in the sn-2 position, and a zwitterionic phosphocholine headgroup in the sn-1 position of a glycerol moiety. The long C32 alkyl chain is terminated either by a second phosphocholine (PC-Gly(2C16Me)C32-PC) or by a phosphodimethylethanolamine headgroup (PC-Gly(2C16Me)C32-Me2PE). The temperature- and pH-dependent aggregation behavior of both lipids was studied using differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS) experiments. The morphology of the formed aggregates in an aqueous suspension was visualized by transmission electron microscopy (TEM). We show that PC-Gly(2C16Me)C32-PC and PC-Gly(2C16Me)C32-Me2PE at pH 5 self-assemble into large lamellar aggregates and large lipid vesicles. Within these structures, the bolalipid molecules are probably assembled in a monolayer with fully interdigitated chains. The lipid molecules seem to be tilted with respect to the layer normal to ensure a dense packing of the alkyl chains. A temperature increase leads to a transition from a lamellar gel phase to the liquid-crystalline phase at about 28-30 °C for both bolalipids. The lamellar aggregates of PC-Gly(2C16Me)C32-Me2PE started to transform into nanofibers when the pH value of the suspension was increased to above 11. At pH 12, these nanofibers were the dominant aggregates.

Azide-Modified Membrane Lipids: Synthesis, Properties, and Reactivity.

In the present work, we describe the synthesis and the temperature-dependent behavior of photoreactive membrane lipids as well as their capability to study peptide/lipid interactions. The modified phospholipids contain an azide group either in the middle part or at the end of an alkyl chain and also differ in the linkage (ester vs ether) of the second alkyl chain. The temperature-dependent aggregation behavior of the azidolipids was studied using differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small-angle X-ray scattering (SAXS). Aggregate structures were visualized by stain and cryo transmission electron microscopy (TEM) and were further characterized by dynamic light scattering (DLS). We show that the position of the azide group and the type of linkage of the alkyl chain at the sn-2 position of the glycerol influences the type of aggregates formed as well as their long-term stability: P10AzSPC and r12AzSHPC show the formation of extrudable liposomes, which are stable in size during storage. In contrast, azidolipids that carry a terminal azido moiety either form extrudable liposomes, which show time-dependent vesicle fusion (P15AzPdPC), or self-assemble in large sheet-like, nonextrudable aggregates (r15AzPdHPC) where the lipid molecules are arranged in an interdigitated orientation at temperatures below Tm (LßI phase). Finally, a P10AzSPC:DMPC mixture was used for photochemically induced cross-linking experiments with a transmembrane peptide (WAL-peptide) to demonstrate the applicability of the azidolipids for the analysis of peptide/lipid interactions. The efficiency of photo-cross-linking was monitored by attenuated total reflection infrared (ATR-IR) spectroscopy and mass spectrometry (MS).

Hierarchical Micelles via Polyphilic Interactions: Hydrogen-Bonded Supramolecular Dendrons and Double Immiscible Polymers.

We report a simple strategy to form three-phase segregated hierarchical micelles via a counterbalanced phase segregation/self-assembly process. Our methodology relies on a cooperative polyphilic phase segregation, paralleled by a self-assembly process induced by hydrogen-bonds to afford the generation of supramolecular multicompartment dendrons. The versatile preparation of such hierarchical morphologies is evidenced on the basis of a series of supramolecular dendrons, composed of semifluorinated copolymers, homopolymers, or nonfluorinated polymers. We do have designed and prepared mid- and α,ω-barbiturate (Ba) functionalized poly(n-butyl acrylates), Ba-(PnBuA-Ba)2, together with a series of heterocomplementary α,ω-Hamilton wedge (HW) functionalized polymers via reversible addition-fragmentation chain transfer (co)polymerization. To enable subtle phase segregation processes, the semifluorinated homo- and copolymers HW-P(nBuA-co-PFPA)-HW (prepared via copolymerization of nBuA with 2,2,3,3,3-pentafluoropropyl acrylate (PFPA)) and HW-PPFPA-HW, as well as the nonfluorinated polymer HW-PnBuA-HW and HW-PI-HW (PI, polyisoprene), have been generated. Selective intermolecular complexation between Ba-(PnBuA-Ba)2 and the complementary polymers (such as HW-P(nBuA-co-PFPA)-HW, HW-PPFPA-HW or HW-PI-HW) leads to the successful generation of supramolecular dendrons as evidenced by (1)H NMR and diffusion-ordered NMR spectroscopy, together with the formation of well-defined disc-like nano-objects as demonstrated by microscopy investigations. Transmission electron microscopy demonstrates a unique, uncommon phase behavior showing remarkable three-phase segregated hierarchical micelles, indicative of the desired micellar multicompartments.

Solubilization of Membrane Proteins into Functional Lipid-Bilayer Nanodiscs Using a Diisobutylene/Maleic Acid Copolymer.

Once removed from their natural environment, membrane proteins depend on membrane-mimetic systems to retain their native structures and functions. To this end, lipid-bilayer nanodiscs that are bounded by scaffold proteins or amphiphilic polymers such as styrene/maleic acid (SMA) copolymers have been introduced as alternatives to detergent micelles and liposomes for in vitro membrane-protein research. Herein, we show that an alternating diisobutylene/maleic acid (DIBMA) copolymer shows equal performance to SMA in solubilizing phospholipids, stabilizes an integral membrane enzyme in functional bilayer nanodiscs, and extracts proteins of various sizes directly from cellular membranes. Unlike aromatic SMA, aliphatic DIBMA has only a mild effect on lipid acyl-chain order, does not interfere with optical spectroscopy in the far-UV range, and does not precipitate in the presence of low millimolar concentrations of divalent cations.

Aggregation behaviour of a single-chain, phenylene-modified bolalipid and its miscibility with classical phospholipids.

In the present work, we describe the synthesis of a single-chain, phenylene-modified bolalipid with two phosphocholine headgroups, PC-C18pPhC18-PC, using a Sonogashira cross-coupling reaction as a key step. The aggregation behaviour was studied as a function of temperature using transmission electron microscopy (TEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and small angle neutron scattering (SANS). We show that our new bolalipid self-assembles into nanofibres, which transform into flexible nanofibres at 27 °C and further to small elongated micelles at 45 °C. Furthermore, the miscibility of the bolalipid with bilayer-forming phosphatidylcholines (DMPC, DPPC, and DSPC) was investigated by means of DSC, TEM, FTIR, and small angle X-ray scattering (SAXS). We could show that the PC-C18pPhC18-PC is partially miscible with saturated phosphatidylcholines; however, closed lipid vesicles with an increased thermal stability were not found. Instead, bilayer fragments and disk-like aggregates are formed.