Myr-Arf1 conformational flexibility at the membrane surface sheds light on the interactions with ArfGAP ASAP1.

ADP-ribosylation factor 1 (Arf1) interacts with multiple cellular partners and membranes to regulate intracellular traffic, organelle structure and actin dynamics. Defining the dynamic conformational landscape of Arf1 in its active form, when bound to the membrane, is of high functional relevance and key to understanding how Arf1 can alter diverse cellular processes. Through concerted application of nuclear magnetic resonance (NMR), neutron reflectometry (NR) and molecular dynamics (MD) simulations, we show that, while Arf1 is anchored to the membrane through its N-terminal myristoylated amphipathic helix, the G domain explores a large conformational space, existing in a dynamic equilibrium between membrane-associated and membrane-distal conformations. These configurational dynamics expose different interfaces for interaction with effectors. Interaction with the Pleckstrin homology domain of ASAP1, an Arf-GTPase activating protein (ArfGAP), restricts motions of the G domain to lock it in what seems to be a conformation exposing functionally relevant regions.

Inhibition of the Fibrillation of Amyloid Aß1-40 by Hybrid-Lipid-Polymer Vesicles.

The transformation of functional proteins into amyloidic plaques is responsible for the impairment of neurological functions in patients fallen victim to debilitating neurological conditions like Alzheimer's, Parkinson's, and Huntington's diseases. The nucleating role of amyloid beta (Aß1-40 ) peptide into amyloids is well established. Herein, lipid hybrid-vesicles are generated with glycerol/cholesterol-bearing polymers aiming to alter the nucleation process and modulate the early phases of Aß1-40 fibrillation. Hybrid-vesicles (±100 nm) are prepared by incorporating variable amounts of cholesterol-/glycerol-conjugated poly(di(ethylene glycol)m acrylates)n polymers into 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes. The in vitro fibrillation kinetics coupled to transmission electron microscopy (TEM) is employed to investigate the role of hybrid-vesicles on Aß1-40 fibrillation without destroying the vesicular membrane. Both polymers, when embedded in hybrid-vesicles (up to 20%) significantly prolonged the fibrillation lag phase (tlag ) compared to a small acceleration in the presence of DOPC vesicles, irrespective of the amount of polymers inside the hybrid-vesicles. Along with this notable retardation effect, a morphological transformation of the amyloid's secondary structures to amorphous aggregates or the absence of fibrillar structures when interacting with the hybrid-vesicles is confirmed by TEM and circular dichroism (CD) spectroscopy.

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes.

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P2 effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P2-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P2. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P2 in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P2 in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P2 functions to coordinate cytoskeletal dynamics and secretion.

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.

Giant Endoplasmic Reticulum vesicles (GERVs), a novel model membrane tool.

Artificial giant vesicles have proven highly useful as membrane models in a large variety of biophysical and biochemical studies. They feature accessibility for manipulation and detection, but lack the compositional complexity needed to reconstitute complicated cellular processes. For the plasma membrane (PM), this gap was bridged by the establishment of giant PM vesicles (GPMVs). These native membranes have facilitated studies of protein and lipid diffusion, protein interactions, electrophysiology, fluorescence analysis of lateral domain formation and protein and lipid partitioning as well as mechanical membrane properties and remodeling. The endoplasmic reticulum (ER) is key to a plethora of biological processes in any eukaryotic cell. However, its intracellular location and dynamic and intricate tubular morphology makes it experimentally even less accessible than the PM. A model membrane, which will allow the afore-mentioned types of studies on GPMVs to be performed on ER membranes outside the cell, is therefore genuinely needed. Here, we introduce the formation of giant ER vesicles, termed GERVs, as a new tool for biochemistry and biophysics. To obtain GERVs, we have isolated ER membranes from Saccharomyces cerevisiae and fused them by exploiting the atlastin-like fusion protein Sey1p. We demonstrate the production of GERVs and their utility for further studies.

A Quantitative and Reliable Calibration Standard for Dual-Color Fluorescence Cross-Correlation Spectroscopy.

Dual-color Fluorescence Cross-Correlation Spectroscopy (dcFCCS) allows binding analysis of biomolecules. Combining cross- and autocorrelation amplitudes yields binding degrees and concentrations of bound and unbound species. However, non-ideal detection volume overlap reduces the cross-correlation, causing overestimation of the Kd . The overlap quality factor that relates measured and true cross-correlation amplitudes has been difficult to determine, because neither a perfect 1 : 1 labeled sample nor perfectly overlapping volumes are readily accomplished. Here, we describe how a stochastically labeled sample can be used for quantitative calibration. Lipid vesicles doped with green and red fluorescent dyes yield highly reproducible relative cross-correlations and allow determination of the setup-dependent overlap quality factor. This reliable, affordable and quick-to-prepare calibration standard expedites any quantitative co-localization or binding analysis by dcFCCS.

Controlling the Miscibility of X-Shaped Bolapolyphiles in Lipid Membranes by Varying the Chemical Structure and Size of the Polyphile Polar Headgroup.

Bolaamphiphiles are well-known naturally occurring structures that can increase the thermal and mechanical stability of the phospholipid membrane by incorporation in a transmembrane manner. Modifications of bolaamphiphiles to introduce particular structural elements such as a conjugated aromatic backbone and lateral side chains in the hydrophobic region lead to bolapolyphiles (BPs). We investigated the ability of BPs to form lyotropic phases in water. The BPs had an identical backbone and side chains, but different headgroup structures, leading to different abilities to act as hydrogen bond donors and acceptors. BPs with hydrophilic headgroups capable of acting as hydrogen bond donors as well as acceptors did not form lyotropic phases and were insoluble in water, independent of whether the polar groups were small or large. The extended lipophilic core structure and the multiple intermolecular hydrogen bonds between the headgroups prevented the formation of well-hydrated lyotropic aggregates. A BP with two large hydrophilic headgroups of several ethylene oxide moieties terminated by methyl groups formed sheet- and vesicle-like aggregates in water. These headgroups act only as hydrogen bond acceptors and cannot form hydrogen bonds in the absence of water. The miscibility of BPs with vesicles of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) in water and the resulting aggregate structures were also investigated. For BPs with headgroups acting as donors and acceptors of hydrogen bonds, macroscopic phase separation occurred in the mixed membranes, and two different membrane domains, a DPPC-rich one containing only little polyphile and a BP-rich one containing varying amounts of lipid, were formed. For headgroups without the ability to act as hydrogen bond donors, small BP aggregates were formed that were homogeneously distributed over the membrane. The lateral organization of BPs in lipid membranes can thus be controlled by the nature of the BP headgroup.

A conserved motif promotes HpaB-regulated export of type III effectors from Xanthomonas.

The type III secretion (T3S) system, an essential pathogenicity factor in most Gram-negative plant-pathogenic bacteria, injects bacterial effector proteins directly into the plant cell cytosol. Here, the type III effectors (T3Es) manipulate host cell processes to suppress defence and establish appropriate conditions for bacterial multiplication in the intercellular spaces of the plant tissue. T3E export depends on a secretion signal which is also present in 'non-effectors'. The latter are secreted extracellular components of the T3S apparatus, but are not translocated into the plant cell. How the T3S system discriminates between T3Es and non-effectors is still enigmatic. Previously, we have identified a putative translocation motif (TrM) in several T3Es from Xanthomonas campestris pv. vesicatoria (Xcv). Here, we analysed the TrM of the Xcv effector XopB in detail. Mutation studies showed that the proline/arginine-rich motif is required for efficient type III-dependent secretion and translocation of XopB and determines the dependence of XopB transport on the general T3S chaperone HpaB. Similar results were obtained for other effectors from Xcv. As the arginine residues of the TrM mediate specific binding of XopB to cardiolipin, one of the major lipid components in Xanthomonas membranes, we assume that the association of T3Es to the bacterial membrane prior to secretion supports type III-dependent export.

Measuring Protein Binding to Lipid Vesicles by Fluorescence Cross-Correlation Spectroscopy.

Fluorescence correlation spectroscopy has been previously used to investigate peptide and protein binding to lipid membranes, as it allows for very low amounts of sample, short measurement times and equilibrium binding conditions. Labeling only one of the binding partners, however, comes with certain drawbacks, as it relies on identifying binding events by a change in diffusion coefficient. Since peptide and protein aggregation can obscure specific binding, and since non-stoichiometric binding necessitates the explicit choice of a statistical distribution for the number of bound ligands, we additionally label the liposomes and perform dual-color fluorescence cross-correlation spectroscopy (dcFCCS). We develop a theoretical framework showing that dcFCCS amplitudes allow calculation of the degree of ligand binding and the concentration of unbound ligand, leading to a model-independent binding curve. As the degree of labeling of the ligands does not factor into the measured quantities, it is permissible to mix labeled and unlabeled ligand, thereby extending the range of usable protein concentrations and accessible dissociation constants, KD. The total protein concentration, but not the fraction of labeled protein, needs to be known. In this work, we apply our dcFCCS analysis scheme to Sar1p, a protein of the COPII complex, which binds "major-minor-mix" liposomes. A Langmuir isotherm model yields KD=(2.1±1.1)µM as the single-site dissociation constant. The dcFCCS framework presented here is highly versatile for biophysical analysis of binding interactions. It may be applied to many types of fluorescently labeled ligands and small diffusing particles, including nanodiscs and liposomes containing membrane protein receptors.

Influence of thylakoid membrane lipids on the structure of aggregated light-harvesting complexes of the diatom Thalassiosira pseudonana and the green alga Mantoniella squamata.

The study investigated the effect of the thylakoid membrane lipids monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) on the structure of two algal light-harvesting complexes (LHCs). In contrast to higher plants whose thylakoid membranes are characterized by an enrichment of the neutral galactolipids MGDG and DGDG, both the green alga Mantoniella squamata and the centric diatom Thalassiosira pseudonana contain membranes with a high content of the negatively charged lipids SQDG and PG. The algal thylakoids do not show the typical grana-stroma differentiation of higher plants but a regular arrangement. To analyze the effect of the membrane lipids, the fucoxanthin chlorophyll protein (FCP) complex of T. pseudonana and the LHC of M. squamata (MLHC) were prepared by successive cation precipitation using Triton X-100 as detergent. With this method, it is possible to isolate LHCs with a reduced amount of associated lipids in an aggregated state. The results from 77 K fluorescence and photon correlation spectroscopy show that neither the neutral galactolipids nor the negatively charged lipids are able to significantly alter the aggregation state of the FCP or the MLHC. This is in contrast to higher plants where SQDG and PG lead to a strong disaggregation of the LHCII whereas MGDG and DGDG induce the formation of large macroaggregates. The results indicate that LHCs which are integrated into thylakoid membranes with a high amount of negatively charged lipids and a regular arrangement are less sensitive to lipid-induced structural alterations than their counterparts in membranes enriched in neutral lipids with a grana-stroma differentiation.

Effects of Lateral and Terminal Chains of X-Shaped Bolapolyphiles with Oligo(phenylene ethynylene) Cores on Self-Assembly Behaviour. Part 1: Transition between Amphiphilic and Polyphilic Self-Assembly in the Bulk.

Polyphilic self-assembly leads to compartmentalization of space and development of complex structures in soft matter on different length scales, reaching from the morphologies of block copolymers to the liquid crystalline (LC) phases of small molecules. Whereas block copolymers are known to form membranes and interact with phospholipid bilayers, liquid crystals have been less investigated in this respect. Here, series of bolapolyphilic X-shaped molecules were synthesized and investigated with respect to the effect of molecular structural parameters on the formation of LC phases (part 1), and on domain formation in phospholipid bilayer membranes (part 2). The investigated bolapolyphiles are based on a rod-like π-conjugated oligo(phenylene ethynylene) (OPE) core with two glycerol groups being either directly attached or separated by additional ethylene oxide (EO) units to both ends. The X-shape is provided by two lateral alkyl chains attached at opposite sides of the OPE core, being either linear, branched, or semiperfluorinated. In this report, the focus is on the transition from polyphilic (triphilic or tetraphilic) to binary amphiphilic self-assembly. Polyphilic self-assembly, i.e., segregation of all three or four incorporated units into separate nano-compartments, leads to the formation of hexagonal columnar LC phases, representing triangular honeycombs. A continuous transition from the well-defined triangular honeycomb structures to simple hexagonal columnar phases, dominated by the arrangement of polar columns on a hexagonal lattice in a mixed continuum formed by the lipophilic chains and the OPE rods, i.e., to amphiphilic self-assembly, was observed by reducing the length and volume of the lateral alkyl chains. A similar transition was found upon increasing the length of the EO units involved in the polar groups. If the lateral alkyl chains are enlarged or replaced by semiperfluorinated chains, then the segregation of lateral chains and rod-like cores is retained, even for enlarged polar groups, i.e., the transition from polyphilic to amphiphilic self-assembly is suppressed.

Effects of Lateral and Terminal Chains of X-Shaped Bolapolyphiles with Oligo(phenylene ethynylene) Cores on Self-Assembly Behavior. Part 2: Domain Formation by Self-Assembly in Lipid Bilayer Membranes.

Supramolecular self-assembly of membrane constituents within a phospholipid bilayer creates complex functional platforms in biological cells that operate in intracellular signaling, trafficking and membrane remodeling. Synthetic polyphilic compounds of macromolecular or small size can be incorporated into artificial phospholipid bilayers. Featuring three or four moieties of different philicities, they reach beyond ordinary amphiphilicity and open up avenues to new functions and interaction concepts. Here, we have incorporated a series of X-shaped bolapolyphiles into DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) bilayers of giant unilamellar vesicles. The bolapolyphiles consist of a rod-like oligo(phenylene ethynylene) (OPE) core, hydrophilic glycerol-based headgroups with or without oligo(ethylene oxide) expansions at both ends and two lateral alkyl chains attached near the center of the OPE core. In the absence of DPPC and water, the compounds showed thermotropic liquid-crystalline behavior with a transition between polyphilic and amphiphilic assembly (see part 1 in this issue). In DPPC membranes, various trends in the domain morphologies were observed upon structure variations, which entailed branched alkyl chains of various sizes, alkyl chain semiperfluorination and size expansion of the headgroups. Observed effects on domain morphology are interpreted in the context of the bulk behavior (part 1) and of a model that was previously developed based on spectroscopic and physicochemical data.

Binding of the GTPase Sar1 to a Lipid Membrane Monolayer: Insertion and Orientation Studied by Infrared Reflection⁻Absorption Spectroscopy.

Membrane-interacting proteins are polyphilic polymers that engage in dynamic protein⁻protein and protein⁻lipid interactions while undergoing changes in conformation, orientation and binding interfaces. Predicting the sites of interactions between such polypeptides and phospholipid membranes is still a challenge. One example is the small eukaryotic GTPase Sar1, which functions in phospholipid bilayer remodeling and vesicle formation as part of the multimeric coat protein complex (COPII). The membrane interaction of Sar1 is strongly dependent on its N-terminal 23 amino acids. By monolayer adsorption experiments and infrared reflection-absorption spectroscopy (IRRAS), we elucidate the role of lipids in inducing the amphipathicity of this N-terminal stretch, which inserts into the monolayer as an amphipathic helix (AH). The AH inserting angle is determined and is consistent with the philicities and spatial distribution of the amino acid monomers. Using an advanced method of IRRAS data evaluation, the orientation of Sar1 with respect to the lipid layer prior to the recruitment of further COPII proteins is determined. The result indicates that only a slight reorientation of the membrane-bound Sar1 is needed to allow coat assembly. The time-course of the IRRAS analysis corroborates a role of slow GTP hydrolysis in Sar1 desorption from the membrane.

Self-Assembly of X-Shaped Bolapolyphiles in Lipid Membranes: Solid-State NMR Investigations.

A novel class of rigid-rod bolapolyphilic molecules with three philicities (rigid aromatic core, mobile aliphatic side chains, polar end groups) has recently been demonstrated to incorporate into and span lipid membranes, and to exhibit a rich variety of self-organization modes, including macroscopically ordered snowflake structures with 6-fold symmetry. In order to support a structural model and to better understand the self-organization on a molecular scale, we here report on proton and carbon-13 high-resolution magic-angle spinning solid-state NMR investigations of two different bolapolyphiles (BPs) in model membranes of two different phospholipids (DPPC, DOPC). We elucidate the changes in molecular dynamics associated with three new phase transitions detected by calorimetry in composite membranes of different composition, namely, a change in π-π-packing, the melting of lipid tails associated with the superstructure, and the dissolution and onset of free rotation of the BPs. We derive dynamic order parameters associated with different H-H and C-H bond directions of the BPs, demonstrating that the aromatic cores are well packed below the final phase transition, showing only 180° flips of the phenyl ring, and that they perform free rotations with additional oscillations of the long axis when dissolved in the fluid membrane. Our data suggests that BPs not only form ordered superstructures, but also rather homogeneously dispersed π-packed filaments within the lipid gel phase, thus reducing the corrugation of large vesicles.

Dendritic domains with hexagonal symmetry formed by x-shaped bolapolyphiles in lipid membranes.

A novel class of bolapolyphile (BP) molecules are shown to integrate into phospholipid bilayers and self-assemble into unique sixfold symmetric domains of snowflake-like dendritic shapes. The BPs comprise three philicities: a lipophilic, rigid, π-π stacking core; two flexible lipophilic side chains; and two hydrophilic, hydrogen-bonding head groups. Confocal microscopy, differential scanning calorimetry, XRD, and solid-state NMR spectroscopy confirm BP-rich domains with transmembrane-oriented BPs and three to four lipid molecules per BP. Both species remain well organized even above the main 1,2-dipalmitoyl-sn-glycero-3-phosphocholine transition. The BP molecules only dissolve in the fluid membrane above 70 °C. Structural variations of the BP demonstrate that head-group hydrogen bonding is a prerequisite for domain formation. Independent of the head group, the BPs reduce membrane corrugation. In conclusion, the BPs form nanofilaments by π stacking of aromatic cores, which reduce membrane corrugation and possibly fuse into a hexagonal network in the dendritic domains.

Temperature-dependent in-plane structure formation of an X-shaped bolapolyphile within lipid bilayers.

Polyphilic compound B12 is an X-shaped molecule with a stiff aromatic core, flexible aliphatic side chains, and hydrophilic end groups. Forming a thermotropic triangular honeycomb phase in the bulk between 177 and 182 °C but no lyotropic phases, it is designed to fit into DPPC or DMPC lipid bilayers, in which it phase separates at room temperature, as observed in giant unilamellar vesicles (GUVs) by fluorescence microscopy. TEM investigations of bilayer aggregates support the incorporation of B12 into intact membranes. The temperature-dependent behavior of the mixed samples was followed by differential scanning calorimetry (DSC), FT-IR spectroscopy, fluorescence spectroscopy, and X-ray scattering. DSC results support in-membrane phase separation, where a reduced main transition and new B12-related transitions indicate the incorporation of lipids into the B12-rich phase. The phase separation was confirmed by X-ray scattering, where two different lamellar repeat distances are visible over a wide temperature range. Polarized ATR-FTIR and fluorescence anisotropy experiments support the transmembrane orientation of B12, and FT-IR spectra further prove a stepwise "melting" of the lipid chains. The data suggest that in the B12-rich domains the DPPC chains are still rigid and the B12 molecules interact with each other via π-π interactions. All results obtained at temperatures above 75 °C confirm the formation of a single, homogeneously mixed phase with freely mobile B12 molecules.

Fluorescence correlation spectroscopy: principles and applications.

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.

Insights from reconstitution reactions of COPII vesicle formation using pure components and low mechanical perturbation.

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided.

Binding of amphiphilic and triphilic block copolymers to lipid model membranes: the role of perfluorinated moieties.

A novel class of symmetric amphi- and triphilic (hydrophilic, lipophilic, fluorophilic) block copolymers has been investigated with respect to their interactions with lipid membranes. The amphiphilic triblock copolymer has the structure PGMA(20)-PPO(34)-PGMA(20) (GP) and it becomes triphilic after attaching perfluoroalkyl moieties (F9) to either end which leads to F(9)-PGMA(20)-PPO(34)-PGMA(20)-F(9) (F-GP). The hydrophobic poly(propylene oxide) (PPO) block is sufficiently long to span a lipid bilayer. The poly(glycerol monomethacrylate) (PGMA) blocks have a high propensity for hydrogen bonding. The hydrophobic and lipophobic perfluoroalkyl moieties have the tendency to phase segregate in aqueous as well as in hydrocarbon environments. We performed differential scanning calorimetry (DSC) measurements on polymer bound lipid vesicles under systematic variation of the bilayer thickness, the nature of the lipid headgroup, and the polymer concentration. The vesicles were composed of phosphatidylcholines (DMPC, DPPC, DAPC, DSPC) or phosphatidylethanolamines (DMPE, DPPE, POPE). We showed that GP as well as F-GP binding have membrane stabilizing and destabilizing components. PPO and F9 blocks insert into the hydrophobic part of the membrane concomitantly with PGMA block adsorption to the lipid headgroup layer. The F9 chains act as additional membrane anchors. The insertion of the PPO blocks of both GP and F-GP could be proven by 2D-NOESY NMR spectroscopy. By fluorescence microscopy we show that F-GP binding increases the porosity of POPC giant unilamellar vesicles (GUVs), allowing the influx of water soluble dyes as well as the translocation of the complete triphilic polymer and its accumulation at the GUV surface. These results open a new route for the rational design of membrane systems with specific properties.