Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

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.

Shaping membrane interfaces in lipid vesicles mimicking the cytoplasmic leaflet of myelin through variation of cholesterol and myelin basic protein contents.

Myelin basic protein (MBP) is an intrinsically disordered protein and in the central nervous system (CNS) mainly responsible for connecting the cytoplasmic surfaces of the multilamellar, compact myelin. Increased posttranslational modification of MBP is linked to both, the natural development (from adolescent to adult brains) of myelin, and features of multiple sclerosis. Here, we study how a combination of this intrinsically disordered myelin protein with varying the natural cholesterol content may alter the characteristics of myelin-like membranes and interactions between these membranes. Large unilamellar vesicles (LUVs) with a composition mimicking the cytoplasmic leaflet of myelin were chosen as the model system, in which different parameters contributing to the interactions between the lipid membrane and MBP were investigated. While we use cryo-transmission electron microscopy (TEM) for imaging, dynamic light scattering (DLS) and electrophoretic measurements through continuously-monitored phase-analysis light scattering (cmPALS) were used for a more global overview of particle size and charge, and electron paramagnetic resonance (EPR) spectroscopy was utilized for local behavior of lipids in the vesicles' membranes in aqueous solution. The cholesterol content was varied from 060 % in these LUVs and measurements were performed in the presence and absence of MBP. We find that the composition of the lipid layers is relevant to the interaction with MBP. Not only the size, the shape and the aggregation behavior of the vesicles depend on the cholesterol content, but also within each membrane, cholesterol's freedom of movement, its environmental polarity and its distribution were found to depend on the content using the EPR-active spin-labeled cholesterol (CSOSL). In addition, DLS and EPR measurements probing the transition temperatures of the lipid phases allow a correlation of specific behavior with the human body temperature of 37 °C. Overall, our results aid in understanding the importance of the native cholesterol content in the healthy myelin membrane, which serves as the basis for stable and optimum protein-bilayer interactions. Although studied in this specific myelin-like system, from a more general and materials science-oriented point of view, we could establish how membrane and vesicle properties depend on cholesterol and/or MBP content, which might be useful generally when specific membrane and vesicle characteristics are sought for.

Zwitterionic fluorinated detergents: From design to membrane protein applications.

We report herein the synthesis of zwitterionic sulfobetaine (SB) and dimethylamine oxide (AO) detergents whose alkyl chain is made of either a perfluorohexyl (F6H3) or a perfluoropentyl (F5H5) group linked to a hydrogenated spacer arm. In aqueous solution, the critical micellar concentrations (CMCs) measured by surface tensiometry (SFT) and isothermal titration calorimetry (ITC) were found in the millimolar range (1.3-2.4 mM). The morphologies of the aggregates were evaluated by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM), demonstrating that the two perfluoropentyl derivatives formed small micelles less than 10 nm in diameter, whereas the perfluorohexyl derivatives formed larger and more heterogeneous micelles. The two SB detergents were able to solubilize synthetic lipid vesicles in a few hours; by contrast, the perfluoropentyl AO induced much faster solubilization, whereas the perfluorohexyl AO did not show any solubilization. All detergents were tested for their abilities to stabilize three membrane proteins, namely, bacteriorhodopsin (bR), the Bacillus subtilis ABC transporter BmrA, and the Streptococcus pneumoniae enzyme SpNOX. The SB detergents outperformed the AO derivatives as well as their hydrogenated analogs in stabilizing these proteins. Among the four new compounds, F5H5SB combines many desirable properties for membrane-protein study, as it is a powerful yet gentle detergent.

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.

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.

Azide- and diazirine-modified membrane lipids: Physicochemistry and applicability to study peptide/lipid interactions via cross-linking/mass spectrometry.

Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs.

Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nanodiscs.

Selection and Incorporation of siRNA Carrying Non-Viral Vector for Sustained Delivery from Gellan Gum Hydrogels.

The local controlled release of siRNA is an attractive and rational strategy to enhance and extend the effectiveness of gene therapy. Since naked and unmodified siRNA has a limited cell uptake and knockdown efficiency, the complexation of siRNA with non-viral carriers is often necessary for the delivery of bioactive RNA. We evaluated the performance of three different non-viral siRNA carriers, including DOTAP lipoplexes (DL), chitosan polyplexes (CP), and solid lipid complexes (SLC). The physicochemical properties of the siRNA-nanocarriers were characterized by dynamic light scattering and gel electrophoresis. After in vitro characterization, the carrier with the most appropriate properties was found to be the DL suspension, which was subsequently loaded into a gellan gum hydrogel matrix and examined for its drug load, stability, and homogeneity. The hydrogels microstructure was investigated by rheology to assess the impact of the rheological properties on the release of the siRNA nanocarriers. A controlled release of complexed siRNA over 60 days in vitro was observed. By comparing the results from fluorescence imaging with data received from HPLC measurements, fluorescence imaging was found to be an appropriate tool to measure the release of siRNA complexes. Finally, the bioactivity of the siRNA released from hydrogel was tested and compared to free DL for its ability to knockdown the GFP expression in a DLD1 colon cancer cell model. The results indicate controlled release properties and activity of the released siRNA. In conclusion, the developed formulation is a promising system to provide local controlled release of siRNA over several weeks.

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.

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge.

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios.

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.

Towards the Development of Long Circulating Phosphatidylserine (PS)- and Phosphatidylglycerol (PG)-Enriched Anti-Inflammatory Liposomes: Is PEGylation Effective?

The anionic phospholipids (PLs) phosphatidylserine (PS) and phosphatidylglycerol (PG) are endogenous phospholipids with anti-inflammatory and immunomodulatory activity. A potential clinical use requires well-defined systems and for several applications, a long circulation time is desirable. Therefore, we aimed the development of long circulating liposomes with intrinsic anti-inflammatory activity. Hence, PS- and PG-enriched liposomes were produced, whilst phosphatidylcholine (PC) liposomes served as control. Liposomes were either formulated as conventional or PEGylated formulations. They had diameters below 150 nm, narrow size distributions and composition-dependent surface charges. Pharmacokinetics were assessed non-invasively via in vivo fluorescence imaging (FI) and ex vivo in excised organs over 2 days. PC liposomes, conventionally formulated, were rapidly cleared from the circulation, while PEGylation resulted in prolongation of liposome circulation robustly distributing among most organs. In contrast, PS and PG liposomes, both as conventional or PEGylated formulations, were rapidly cleared. Non-PEGylated PS and PG liposomes did accumulate almost exclusively in the liver. In contrast, PEGylated PS and PG liposomes were observed mainly in liver and spleen. In summary, PEGylation of PS and PG liposomes was not effective to prolong the circulation time but caused a higher uptake in the spleen.

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.

Fluorescent spherical mesoporous silica nanoparticles loaded with emodin: Synthesis, cellular uptake and anticancer activity.

The natural product emodin (EO) exhibits anti-inflammatory, antiangiogenesis and antineoplastic properties in vitro and in vivo. Due to its biological properties as well as its fluorescence, EO can be useful in pharmacology and pharmacokinetics. To enhance its selectivity to cancer cells, EO was loaded into non-fluorescent and novel fluorescent spherical mesoporous nanoparticles bearing N-methyl isatoic anhydride (SNM~M) or lissamine rhodamine B sulfonyl moieties (SNM~L). The propylamine functionalized mesoporous silica nanomaterial (SNM) were characterized by powder X-ray diffraction (XRD), nitrogen gas sorption, scanning electron microscopy (SEM), transmission electron microscopy (TEM), fluorescence spectroscopy, thermogravimetric analysis (TGA) and UV spectroscopy. The cytotoxicity of EO-loaded nanoparticles was tested against the human colon carcinoma cell line HT-29. Non-loaded SNM did not affect cell proliferation, whereas those loaded with EO were at least as efficient as EO alone. It could be shown by fluorescence microscopy that the uptake of silica nanomaterial by the tumor cells occurred within 2 h and the release of EO occurred within 48 h of treatment. Flow cytometry and Western blot analysis showed that SNM containing EO induced apoptosis in HT-29 cells.

Investigating conjugated polymer nanoparticle formulations for lateral flow immunoassays.

Lateral flow immunoassays (LFI) are valuable tools for point-of-care testing. However, their sensitivity is limited and can be further improved. Nanoparticles (NP) of conjugated polymers (CPNs), also known as Pdots, are reported to be highly sensitive fluorescent probes, but a direct comparison with conventional colloidal gold-based (Au-NP) LFI using the same antibody-antigen pair is missing to date. Furthermore, the influence of brightness and Stokes shift of CPs on the signal : background ratio (SBR) needs to be evaluated. In this study, we encapsulated two different CPs, poly-(9,9-di-n-octyl-fluorenyl-2,7-diyl) (PDOF) and poly-(2,5-di-hexyloxy-cyanoterephthalylidene) (CN-PPV) in silica shell-crosslinked Pluronic© micelles (Si-NP) and Pdots and investigated the NP brightness with respect to CP loading dose. The brightest formulation of each NP system was conjugated to rabbit IgG as a model antigen and the SBR was investigated in an ELISA-like microplate assay and LFI. Two reference particles, Au-NP and a polystyrene NP (PS-NP) loaded with a small-molecule fluorescent dye were conjugated to IgG and compared to the Si-NP and Pdots. The mass of Pdots required for detection in LFI was at least two orders of magnitude lower than that of Si-NP and the reference NP. The SBR of CN-PPV (moderate brightness, large Stokes shift) was two to three times higher than the SBR of PDOF (high brightness, small Stokes shift). To combine the favourable properties of both CPs, a polymer blend of PDOF and CN-PPV was encapsulated in Pdots, and resulted in further increase of SBR in the microplate assay and LFI. In summary, combining two CPs with different properties can lead to fluorescent signal-transducers for applications such as ELISA and LFIs, which can enhance the detection limit of the assay by 2-3 orders of magnitude.

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.