Functional determinants of a synthetic vesicle fusion system

Selective membrane mergers may be driven by small-molecule recognition between synthetic surface-displayed fusogens which bear vancomycin glycopeptide and its native binding target, D-Ala-D-Ala dipeptide. These recognition motifs are membrane anchored by antimicrobial peptide magainin II and a phosphatidylethanolamine lipid derivative, respectively. We report herein characterization of this synthetic membrane fusion reaction with regard to the following: effects of fusogen concentration, lipid composition, and membrane charge. Our findings indicate that these parameters are determinants of fusion rate, vesicle stability, peptide binding, catalytic fusion and membrane disruption during fusion. Notably, these data indicate the importance of coupling between molecular recognition and insertion for bilayer activation as well as the critical role of membrane subdomain formation for membrane fusion reactivity. These phenomena are general to lipid membrane chemistry, and therefore these findings provide a guideline for understanding more complex biomembrane systems.     

SNARE derived peptide mimic inducing membrane fusion

SNARE proteins mediate membrane fusion between synaptic vesicles and the plasma membrane. A minimized peptide SNARE model system with reduced complexity was introduced combining the native SNARE transmembrane (TMD) and linker domains with artificial coiled-coil forming peptides. Specific membrane fusion initiated by coiled-coil recognition was shown by lipid and content mixing vesicle assays.     

Intra- and intermembrane pairwise molecular recognition between synthetic hydrogen-bonding phospholipids

Multivalency and preorganization are fundamental aspects of molecular recognition at the lipid membrane-water interface and can render weak monomeric binding interactions selective and robust; this concept is important throughout biology, biotechnology, and materials science. Though hydrogen bonding is typically weakened in water, intramembrane hydrogen bonding between native lipids has been well-studied and is thought to contribute to lipid bioactivity and membrane function. We hypothesized that avidity and preorganization effects at the lipid-water interface could overcome solvent competition and allow for selective hydrogen-bond recognition between small, unstructured components. We have found that electrostatically identical vesicular membranes composed of cyanuric acid and melamine functionalized phospholipids 1 and 2 undergo selective apposition, fusion and adhesion in suspension and on solid support, indicating that their well-known low-dielectric hydrogen bonding properties translate effectively to the lipid-water interface. This work is notable and of general interest given the few detailed studies of aqueous phase hydrogen-bonding systems; we have extensively characterized this system, gaining structural, functional, and thermodynamic data. Furthermore, we have found that the designed lipid-lipid headgroup interactions result in dramatic alteration of the lipid phase morphology, providing insight into the coupling of molecular interactions with assembly state. As such, this work contributes to our understanding of fundamental phenomena such as molecular recognition at the lipid-water interface membrane chemistry and further illustrates the general possibility of designing selective hydrogen-bonding adhesive interactions from simple starting materials at other polar-apolar interfaces; this could have numerous materials and biotechnological applications.     

An improved poly(A) motifs recognition method based on decision level fusion

Polyadenylation is the process of addition of poly(A) tail to mRNA 3′ ends. Identification of motifs controlling polyadenylation plays an essential role in improving genome annotation accuracy and better understanding of the mechanisms governing gene regulation. The bioinformatics methods used for poly(A) motifs recognition have demonstrated that information extracted from sequences surrounding the candidate motifs can differentiate true motifs from the false ones greatly. However, these methods depend on either domain features or string kernels. To date, methods combining information from different sources have not been found yet. Here, we proposed an improved poly(A) motifs recognition method by combing different sources based on decision level fusion. First of all, two novel prediction methods was proposed based on support vector machine (SVM): one method is achieved by using the domain-specific features and principle component analysis (PCA) method to eliminate the redundancy (PCA–SVM); the other method is based on Oligo string kernel (Oligo-SVM). Then we proposed a novel machine-learning method for poly(A) motif prediction by marrying four poly(A) motifs recognition methods, including two state-of-the-art methods (Random Forest (RF) and HMM-SVM), and two novel proposed methods (PCA–SVM and Oligo-SVM). A decision level information fusion method was employed to combine the decision values of different classifiers by applying the DS evidence theory. We evaluated our method on a comprehensive poly(A) dataset that consists of 14,740 samples on 12 variants of poly(A) motifs and 2750 samples containing none of these motifs. Our method has achieved accuracy up to 86.13%. Compared with the four classifiers, our evidence theory based method reduces the average error rate by about 30%, 27%, 26% and 16%, respectively. The experimental results suggest that the proposed method is more effective for poly(A) motif recognition.     

Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system.     

Electrophysiological Characterization of Transport Across Outer‐Membrane Channels from Gram‐Negative Bacteria in Presence of Lipopolysaccharides

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we present an approach using fusion of native outer membrane vesicles (OMVs) into a planar lipid bilayer, allowing characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion surprisingly revealed only single or few channel activities. The asymmetry of the OMVs translates after fusion into the lipid membrane with the lipopolysaccharides (LPS) dominantly present at the side of OMV addition. Compared to the conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles for functional and structural studies of membrane channels in the native membrane.     

Experimental evidence to support a theory of lipid membrane fusion

Membrane fusion between two lipid membranes with different curvatures was measured by using a fluorescence fusion assay for lipid vesicle systems and was also obtained by measuring lipid monolayer surface tension upon the fusion of vesicles to monolayer membranes. For such membrane systems, it was found that when lysolipid was incorporated only in the membrane with a greater curvature, membrane fusion was more suppressed than those for the case where the same amount (molar ratio of lysolipid to non-lysolipids) of lysolipid was incorporated only in the membrane with a lower curvature. When lysolipid was incorporated only in a flat membrane (e.g., monolayer) and the fusion of small vesicles (SUV) to the monolayer was measured, suppression of membrane fusion by lysolipid was minimal. It is known that lysolipid lowers the surface energy of curved membranes, which stabilizes energetically such membrane surfaces, and thus suppresses membrane fusion. Our results support our theory of lipid membrane fusion where the membrane fusion occurs through the most curved membrane region at the contact area of two interacting membranes.     

Fusion of lipid vesicles with planar lipid bilayers induced by a combination of peptides

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRF) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRF microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.     

Molecular view of the role of fusion peptides in promoting positive membrane curvature

Fusion peptides are moderately hydrophobic segments of viral and nonviral membrane fusion proteins that enable these proteins to fuse two closely apposed biological membranes. In vitro assays furthermore show that even isolated fusion peptides alone can support membrane fusion in model systems. In addition, the fusion peptides have a distinct effect on the phase diagram of lipid mixtures. Here, we present molecular dynamics simulations investigating the effect of a particular fusion peptide, the influenza hemagglutinin fusion peptide and some of its mutants, on the lipid phase diagram. We detect a systematic shift toward phases with more positive mean curvature in the presence of the peptides, as well as an occurrence of bicontinuous cubic phases, which indicates a stabilization of Gaussian curvature. The wild-type fusion peptide has a stronger effect on the phase behavior as compared to the mutants, which we relate to its boomerang shape. Our results point to a different role of fusion peptides than hitherto assumed, the stabilization of pores rather than stalks along the fusion pathway.     

Interrogating Membrane Protein Conformational Dynamics within Native Lipid Compositions

The interplay between membrane proteins and the lipids of the membrane is important for cellular function, however, tools enabling the interrogation of protein dynamics within native lipid environments are scarce and often invasive. We show that the styrene–maleic acid lipid particle (SMALP) technology can be coupled with hydrogen–deuterium exchange mass spectrometry (HDX‐MS) to investigate membrane protein conformational dynamics within native lipid bilayers. We demonstrate changes in accessibility and dynamics of the rhomboid protease GlpG, captured within three different native lipid compositions, and identify protein regions sensitive to changes in the native lipid environment. Our results illuminate the value of this approach for distinguishing the putative role(s) of the native lipid composition in modulating membrane protein conformational dynamics.     

Photoinactivation of Sindbis Virus Infectivity Without Inhibition of Membrane Fusion

Photoinactivation of enveloped viruses is commonly associated with damage to fusion proteins and inhibition of membrane fusion capacity. Here we show that photobleaching of Sindbis virus labeled with the membrane localized dye, R18 (octadecyl rhodamine B) causes a dramatic loss of infectivity without observable changes in low-pH triggered membrane fusion to liposomes. Sindbis labeled with DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) also maintains low-pH triggered membrane fusion capacity, but in contrast to R18, extensive photobleaching of DiI-labeled virus has little effect on infectivity. Electrophoretic gel analysis suggests no cross-linking of viral fusion proteins following photobleaching of dye-labeled Sindbis. These observations have implications for live-cell, single particle tracking studies of dye-labeled Sindbis virus. Our observations suggest that R18 and DiI have different propensities for spontaneous flip-flop in lipid bilayers.     

Templated assembly of biomembranes on silica microspheres using bacteriorhodopsin conjugates as structural anchors

Membrane proteins are some of the most sophisticated molecules found in nature. These molecules have extraordinary recognition properties; hence, they represent a vast source of specialized materials with potential uses in sensing and screening applications. However, the strict requirement of the native lipid environment to preserve their structure and functionality presents an impediment in building biofunctional materials from these molecules. In general, the purification protocols remove the native lipid support structures found in the cellular environment that stabilize the membrane proteins. Furthermore, the membrane protein structure is often highly complex, typified by large, multisubunit complexes that not only span the lipid bilayer but also contain large (>2 nm) cytoplasmic and extracellular domains that protrude from the membrane. The present study is focused on using a biomimetic approach to build a stable, fluid microenvironment to be used to incorporate larger membrane proteins of interest into a tether-supported lipid bilayer membrane adequately spaced above a substrate passivated to liposome fusion and nonspecific adsorption. Our aim is to reintroduce the supporting structures of the native cell membrane using self-assembled supramolecular complexes constructed on microspheres in an artificial cytoskeleton motif. Central to our architecture is to utilize bacteriorhodopsin (bR), a transmembrane protein, as a biomembrane anchoring molecule to be tethered to surfaces of interest as a sparse structural element in the design. Compared to a typical lipid tether, which inserts into one leaflet of the lipid bilayer, bR anchoring provides an over 8-fold greater hydrophobic surface area in contact with the bilayer. In the work presented here, the silica microsphere surface was biofunctionalized with streptavidin to make it a suitable supporting interface. This was achieved by self-assembly of (p-aminophenyl)trimethoxysilane on the silica surface followed by subsequent conjugation of biotin-PEG3400 (PEG = poly(ethylene glycol) and PEG2000 for further passivation and the binding of streptavidin. We have conjugated bR with biotin-PEG3400 through amine-based coupling to use it as a tether. The biotin-PEG-bR conjugate was further labeled with Texas Red to facilitate localization via fluorescence imaging. Confocal microscopy was utilized to analyze the microsphere surface at different stages of surface modification by employing fluorescent staining techniques. Sparely tethered supported lipid bilayer membranes were constructed successfully on streptavidin-functionalized silica particles (5 mum) using a detergent-based method in which tethered bR nucleates self-assembly of the bilayer membrane. The fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching in confocal imaging detection mode. The phospholipid diffusion coefficients obtained from these studies indicated that nativelike fluidity was achieved in the tether-supported membranes, thus providing a prospective microenvironment for insertion of membrane proteins of interest.     

Directed peptide assembly at the lipid-water interface cooperatively enhances membrane binding and activity

We modified membrane-active peptides with synthetic recognition modules to foster peptide assembly at the lipid-water interface. The designed recognition strategy has been previously reported: tris-cyanuric acid and tris-melamine have been found to bind selectively to each another when membrane-anchored. We designed this interaction to occur between two membrane-active peptides, forming a heteromeric complex at the lipid-water interface that exhibits superior membrane binding and permeation compared to the monomeric peptides, presumably because of the higher avidity of the assembled structure. These conjugates do not assemble appreciably in solution but assemble at the lipid-water interface, with surface binding of the peptide acting cooperatively with molecular recognition to yield improved binding and permeation. Furthermore, we find that specific recognition between tris-cyanuric acid phospholipid (TCA-PE) at low surface concentration and tris-melamine magainin (TMM) or hexa-melamine magainin (HMM) results in highly lytic binding, whereas no binding is detectable in the absence of lipid recognition. These findings suggest a noncovalent strategy to enhance peptide membrane activity, which may lead to the discovery of more potent surface-active agents such as antimicrobials.     

Dynamics of a bilayer membrane with membrane-solvent partial slip boundary conditions

We discuss the dynamics of a bilayer membrane with partial slip boundary conditions between the monolayers and the bulk fluid. Using Onsager’s variational principle to account for the associated dissipations, we derive the coupled dynamic equations for the membrane height and the excess lipid density. The newly introduced friction coe?cients appear in the renormalized fluid viscosities. For ordinary lipid bilayer membranes, we find that it is generally justified to ignore the e?ects of permeation and parallel slip at the membrane surface.     

Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed 15N NMR relaxation

Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR (15)N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein-membrane interactions.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

Fusogenic tilted peptides induce nanoscale holes in supported phosphatidylcholine bilayers

Tilted peptides are known to insert in lipid bilayers with an oblique orientation, thereby destabilizing membranes and facilitating membrane fusion processes. Here, we report the first direct visualization of the interaction of tilted peptides with lipid membranes using in situ atomic force microscopy (AFM) imaging. Phase-separated supported dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers were prepared by fusion of small unilamellar vesicles and imaged in buffer solution, in the absence and in the presence of the simian immunodeficiency virus (SIV) peptide. The SIV peptide was shown to induce the rapid appearance of nanometer scale bilayer holes within the DPPC gel domains, while keeping the domain shape unaltered. We attribute this behavior to a local weakening and destabilization of the DPPC domains due to the oblique insertion of the peptide molecules. These results were directly correlated with the fusogenic activity of the peptide as determined using fluorescently labeled DOPC/DPPC liposomes. By contrast, the nontilted ApoE peptide did not promote liposome fusion and did not induce bilayer holes but caused slight erosion of the DPPC domains. In conclusion, this work provides the first direct evidence for the production of stable, well-defined nanoholes in lipid bilayer domains by the SIV peptide, a behavior that we have shown to be specifically related to the tilted character of the peptide. A molecular mechanism underlying spontaneous insertion of the SIV peptide within lipid bilayers and the subsequent removal of bilayer patches is proposed, and its relevance to membrane fusion processes is discussed.     

Molecular basis for dimethylsulfoxide (DMSO) action on lipid membranes

Dimethylsulfoxide (DMSO) is an aprotic solvent that has the ability to induce cell fusion and cell differentiation and enhance the permeability of lipid membranes. It is also an effective cryoprotectant. Insights into how this molecule modulates membrane structure and function would be invaluable toward regulating the above processes and for developing chemical means for enhancing or hindering the absorption of biologically active molecules, in particular into or via the skin. We show here by means of molecular simulations that DMSO can induce water pores in dipalmitoyl-phosphatidylcholine bilayers and propose this to be a possible pathway for the enhancement of penetration of actives through lipid membranes. DMSO also causes the membrane to become floppier, which would enhance permeability, facilitate membrane fusion, and enable the cell membrane to accommodate osmotic and mechanical stresses during cryopreservation.     

The design of molecular sensing interfaces with lipid-bilayer assemblies

We describe the current status of techniques for preparing planar lipid bilayers, the fusion of sensory elements and the design of bilayer interfaces for analytical applications. Advances in bilayer fabrication have allowed preparation of lipid bilayers with membrane resistance of >1 GΩ (“gigaseal”) in flow, microfluidic and array formats, which have enabled single-channel and multi-channel recording. Not only biological but also engineered channels have been adopted as sensory elements for lipid-bilayer-based biosensors. Synthetic and inorganic channels are also emerging for designing membranes for lipid-bilayer sensors. We discuss the potential of lipid bilayers containing biological, engineered or synthetic channels for the design of biosensors, including drug-screening biosensors.     

Effect of lipid molecule headgroup mismatch on non steroidal anti-inflammatory drugs induced membrane fusion

Membrane fusion is an essential process guiding many important biological events, which most commonly requires the aid of proteins and peptides as fusogenic agents. Small drug induced fusion at low drug concentration is a rare event. Only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs) have been shown by us to induce membrane fusion successfully at low drug concentration. A better elucidation of the mechanism and the effect of different parameters in modulating the fusion process will allow the use of these common drugs to induce and control membrane fusion in various biochemical processes. In this study, we monitor the effect of lipid headgroup size mismatch in the bilayer on oxicam NSAIDs induced membrane fusion, by introducing dimyristoylphosphatidylethanolamine (DMPE) in dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs). Such headgroup mismatch affects various lipid parameters which includes inhibition of trans-bilayer motion, domain formation, decrease in curvature, etc. Changes in various lipidic parameters introduce defects in the membrane bilayer and thereby modulate membrane fusion. SUVs formed by DMPC with increasing DMPE content (10, 20, and 30 mol %) were used as simple model membranes. Transmission electron microscopy (TEM) and differential scanning calorimetry (DSC) were used to characterize the DMPC-DMPE mixed vesicles. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. How the inhibition of trans-bilayer motion, heterogeneous distribution of lipids, decrease in vesicle curvature, etc., arising due to headgroup mismatch affect the fusion process has been isolated and identified here. Mx amplifies these effects maximally followed by Px and Tx. This has been correlated to the enhanced partitioning of the hydrophobic Mx compared to the more hydrophilic Px and Tx in the mixed bilayer.