Stability of a biomembrane tube covered with proteins.

Membrane tubes are essential structural features in cells that facilitate biomaterial transport and inter- and intracellular signaling. The shape of these tubes can be regulated by the proteins that surround and adhere to them. We study the stability of a biomembrane tube coated with proteins by combining linear stability analysis, out-of-equilibrium hydrodynamic calculations, and numerical solutions of a Helfrich-like membrane model. Our analysis demonstrates that both long- and short-wavelength perturbations can destabilize the tubes. Numerical simulations confirm the derived linear stability criteria and yield the nonlinearly perturbed vesicle shapes. Our study highlights the interplay between membrane shape and protein density, where the shape instability concurs with a redistribution of proteins into a banded pattern.

Architecture of the chikungunya virus replication organelle.

Alphaviruses are mosquito-borne viruses that cause serious disease in humans and other mammals. Along with its mosquito vector, the Alphavirus chikungunya virus (CHIKV) has spread explosively in the last 20 years, and there is no approved treatment for chikungunya fever. On the plasma membrane of the infected cell, CHIKV generates dedicated organelles for viral RNA replication, so-called spherules. Whereas structures exist for several viral proteins that make up the spherule, the architecture of the full organelle is unknown. Here, we use cryo-electron tomography to image CHIKV spherules in their cellular context. This reveals that the viral protein nsP1 serves as a base for the assembly of a larger protein complex at the neck of the membrane bud. Biochemical assays show that the viral helicase-protease nsP2, while having no membrane affinity on its own, is recruited to membranes by nsP1. The tomograms further reveal that full-sized spherules contain a single copy of the viral genome in double-stranded form. Finally, we present a mathematical model that explains the membrane remodeling of the spherule in terms of the pressure exerted on the membrane by the polymerizing RNA, which provides a good agreement with the experimental data. The energy released by RNA polymerization is found to be sufficient to remodel the membrane to the characteristic spherule shape.

Endocytosis of Coacervates into Liposomes.

Recent studies have shown that the interactions between condensates and biological membranes are of functional importance. Here, we study how the interaction between complex coacervates and liposomes as model systems can lead to wetting, membrane deformation, and endocytosis. Depending on the interaction strength between coacervates and liposomes, the wetting behavior ranged from nonwetting to engulfment (endocytosis) and complete wetting. Endocytosis of coacervates was found to be a general phenomenon: coacervates made from a wide range of components could be taken up by liposomes. A simple theory taking into account surface energies and coacervate sizes can explain the observed morphologies. Our findings can help to better understand condensate-membrane interactions in cellular systems and provide new avenues for intracellular delivery using coacervates.

Design and Functional Analysis of Heterobifunctional Multivalent Phage Capsid Inhibitors Blocking the Entry of Influenza Virus.

Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qß) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qß conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.

Dielectric constant of aqueous solutions of proteins and organic polymers from molecular dynamics simulations.

The dielectric constant of water/oligomer mixtures, spanning the range from pure water to pure oligomeric melts, is investigated using molecular dynamics (MD) simulations. As prototypical water-soluble organic substances, we consider neutral poly-glycine, poly-ethylene glycol, and charged monomeric propionic acid. As the water content is reduced, the dielectric constant decreases but does not follow an ideal mixing behavior. The deviations from ideal mixing originate primarily in the non-linear relation between the oligomer mass fraction and collective polarization effects. We find that the dielectric constant is dominated by water polarization, even if the oligomer mass fraction exceeds 50%. By a double extrapolation of the MD simulation results to the limit of vanishing water fraction and to the limit of infinite oligomeric chain length, we estimate the orientational contribution to the dielectric constant of the pure polymeric melts. By this procedure, we obtain ɛ = 17 ± 2 for polyglycine and ɛ = 1 ± 0.3 for polyethylene glycol. The large difference is rationalized by polarization correlations of glycine units. Interestingly, we find constant temperature simulations to outperform replica exchange simulations in terms of equilibration speed.

Membrane shape remodeling by protein crowding.

The steric repulsion between proteins on biological membranes is one of the most generic mechanisms that cause membrane shape changes. We present a minimal model in which a spontaneous curvature is induced by asymmetric protein crowding. Our results show that the interplay between the induced spontaneous curvature and the membrane tension determines the energy-minimizing shapes, which describes the wide range of experimentally observed membrane shapes, i.e., flat membranes, spherical vesicles, elongated tubular protrusions, and pearling structures. Moreover, the model gives precise predictions on how membrane shape changes by protein crowding can be tuned by controlling the protein size, the density of proteins, and the size of the crowded domain.

Diffusion on Membrane Domes, Tubes, and Pearling Structures.

Diffusion is a fundamental mechanism for protein distribution in cell membranes. These membranes often exhibit complex shapes, which range from shallow domes to elongated tubular or pearl-like structures. Shape complexity of the membrane influences the diffusive spreading of proteins and molecules. Despite the importance membrane geometry plays in these diffusive processes, it is challenging to establish the dependence between diffusion and membrane morphology. We solve the diffusion equation numerically on various static curved shapes representative for experimentally observed membrane shapes. Our results show that membrane necks become diffusion barriers. We determine the diffusive half-time, i.e., the time that is required to reduce the amount of protein in the budded region by one half, and find a quadratic relation between the diffusive half-time and the averaged mean curvature of the membrane shape, which we rationalize by a scaling law. Our findings thus help estimate the characteristic diffusive timescale based on the simple measure of membrane mean curvature.

Protein crowding mediates membrane remodeling in upstream ESCRT-induced formation of intraluminal vesicles.

As part of the lysosomal degradation pathway, the endosomal sorting complexes required for transport (ESCRT-0 to -III/VPS4) sequester receptors at the endosome and simultaneously deform the membrane to generate intraluminal vesicles (ILVs). Whereas ESCRT-III/VPS4 have an established function in ILV formation, the role of upstream ESCRTs (0 to II) in membrane shape remodeling is not understood. Combining experimental measurements and electron microscopy analysis of ESCRT-III-depleted cells with a mathematical model, we show that upstream ESCRT-induced alteration of the Gaussian bending rigidity and their crowding in concert with the transmembrane cargo on the membrane induce membrane deformation and facilitate ILV formation: Upstream ESCRT-driven budding does not require ATP consumption as only a small energy barrier needs to be overcome. Our model predicts that ESCRTs do not become part of the ILV, but localize with a high density at the membrane neck, where the steep decline in the Gaussian curvature likely triggers ESCRT-III/VPS4 assembly to enable neck constriction and scission.

Diffuso-kinetic membrane budding dynamics.

A wide range of proteins are known to create shape transformations of biological membranes, where the remodelling is a coupling between the energetic costs from deforming the membrane, the recruitment of proteins that induce a local spontaneous curvature C0 and the diffusion of proteins along the membrane. We propose a minimal mathematical model that accounts for these processes to describe the diffuso-kinetic dynamics of membrane budding processes. By deploying numerical simulations we map out the membrane shapes, the time for vesicle formation and the vesicle size as a function of the dimensionless kinetic recruitment parameter K1 and the proteins sensitivity to mean curvature. We derive a time for scission that follows a power law ∼K1-2/3, a consequence of the interplay between the spreading of proteins by diffusion and the kinetic-limited increase of the protein density on the membrane. We also find a scaling law for the vesicle size ∼1/([small sigma, Greek, macron]avC0), with [small sigma, Greek, macron]av the average protein density in the vesicle, which is confirmed in the numerical simulations. Rescaling all the membrane profiles at the time of vesicle formation highlights that the membrane adopts a self-similar shape.

Rapid Growth and Fusion of Protocells in Surface-Adhered Membrane Networks.

Elevated temperatures might have promoted the nucleation, growth, and replication of protocells on the early Earth. Recent reports have shown evidence that moderately high temperatures not only permit protocell assembly at the origin of life, but can have actively supported it. Here, the fast nucleation and growth of vesicular compartments from autonomously formed lipid networks on solid surfaces, induced by a moderate increase in temperature, are shown. Branches of the networks, initially consisting of self-assembled interconnected nanotubes, rapidly swell into microcompartments which can spontaneously encapsulate RNA fragments. The increase in temperature further causes fusion of adjacent network-connected compartments, resulting in the redistribution of the RNA. The experimental observations and the mathematical model indicate that the presence of nanotubular interconnections between protocells facilitates the fusion process.

Quantification of Multivalent Interactions between Sialic Acid and Influenza A Virus Spike Proteins by Single-Molecule Force Spectroscopy.

Multivalency is a key principle in reinforcing reversible molecular interactions through the formation of multiple bonds. The influenza A virus deploys this strategy to bind strongly to cell surface receptors. We performed single-molecule force spectroscopy (SMFS) to investigate the rupture force required to break individual and multiple bonds formed between synthetic sialic acid (SA) receptors and the two principal spike proteins of the influenza A virus (H3N2): hemagglutinin (H3) and neuraminidase (N2). Kinetic parameters such as the rupture length (χß) and dissociation rate (koff) are extracted using the model by Friddle, De Yoreo, and Noy. We found that a monovalent SA receptor binds to N2 with a significantly higher bond lifetime (270 ms) compared to that for H3 (36 ms). By extending the single-bond rupture analysis to a multibond system of n protein-receptor pairs, we provide an unprecedented quantification of the mechanistic features of multivalency between H3 and N2 with SA receptors and show that the stability of the multivalent connection increases with the number of bonds from tens to hundreds of milliseconds. Association rates (kon) are also provided, and an estimation of the dissociation constants (KD) between the SA receptors to both proteins indicate a 17-fold higher binding affinity for the SA-N2 bond with respect to that of SA-H3. An optimal designed multivalent SA receptor showed a higher binding stability to the H3 protein of the influenza A virus than to the monovalent SA receptor. Our study emphasizes the influence of the scaffold on the presentation of receptors during multivalent binding.

Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1-4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle7.

Nanotube-Mediated Path to Protocell Formation.

Cellular compartments are membrane-enclosed, spatially distinct microenvironments that confine and protect biochemical reactions in the biological cell. On the early Earth, the autonomous formation of compartments is thought to have led to the encapsulation of nucleotides, thereby satisfying a starting condition for the emergence of life. Recently, surfaces have come into focus as potential platforms for the self-assembly of prebiotic compartments, as significantly enhanced vesicle formation was reported in the presence of solid interfaces. The detailed mechanism of such formation at the mesoscale is still under discussion. We report here on the spontaneous transformation of solid-surface-adhered lipid deposits to unilamellar membrane compartments through a straightforward sequence of topological changes, proceeding via a network of interconnected lipid nanotubes. We show that this transformation is entirely driven by surface-free energy minimization and does not require hydrolysis of organic molecules or external stimuli such as electrical currents or mechanical agitation. The vesicular structures take up and encapsulate their external environment during formation and can subsequently separate and migrate upon exposure to hydrodynamic flow. This may link the self-directed transition from weakly organized bioamphiphile assemblies on solid surfaces to protocells with secluded internal contents.

Exploring the Limits of Bivalency by DNA-Based Spatial Screening.

Multivalency can facilitate complex formation when monovalent receptor-ligand interactions are weak. However, enhanced binding of two multivalent binding partners should be avoidable, for example when bivalent receptors ought to utilize multimolecular interactions to cross-link binding partners. We herein report the first systematic study to assess the criteria deciding whether a bivalent system engages in bivalency-enhanced interactions or cross-linking. We used DNA-instructed self-assembly to arrange the cucurbit[7]uril-adamantane host-guest system in 70-360 Šdistance. Measurements and statistical mechanics analyses revealed that the affinity gain is controlled by 1) the distance between recognition modules, 2) the scaffold flexibility, and, importantly, 3) the strength of the monovalent interaction. We show that the bivalency effect can extend beyond 150 Šand discuss how, on the contrary, weak monovalent interactions reduce the concentration threshold for cross-linking. The findings are of interest for inhibitor design.

Exploring Rigid and Flexible Core Trivalent Sialosides for Influenza Virus Inhibition.

Herein, the chemical synthesis and binding analysis of functionalizable rigid and flexible core trivalent sialosides bearing oligoethylene glycol (OEG) spacers interacting with spike proteins of influenza A virus (IAV) X31 is described. Although the flexible Tris-based trivalent sialosides achieved micromolar binding constants, a trivalent binder based on a rigid adamantane core dominated flexible tripodal compounds with micromolar binding and hemagglutination inhibition constants. Simulation studies indicated increased conformational penalties for long OEG spacers. Using a systematic approach with molecular modeling and simulations as well as biophysical analysis, these findings emphasize on the importance of the scaffold rigidity and the challenges associated with the spacer length optimization.

Quantitative Prediction of Multivalent Ligand-Receptor Binding Affinities for Influenza, Cholera, and Anthrax Inhibition.

Multivalency achieves strong, yet reversible binding by the simultaneous formation of multiple weak bonds. It is a key interaction principle in biology and promising for the synthesis of high-affinity inhibitors of pathogens. We present a molecular model for the binding affinity of synthetic multivalent ligands onto multivalent receptors consisting of n receptor units arranged on a regular polygon. Ligands consist of a geometrically matching rigid polygonal core to which monovalent ligand units are attached via flexible linker polymers, closely mimicking existing experimental designs. The calculated binding affinities quantitatively agree with experimental studies for cholera toxin ( n = 5) and anthrax receptor ( n = 7) and allow to predict optimal core size and optimal linker length. Maximal binding affinity is achieved for a core that matches the receptor size and for linkers that have an equilibrium end-to-end distance that is slightly longer than the geometric separation between ligand core and receptor sites. Linkers that are longer than optimal are greatly preferable compared to shorter linkers. The angular steric restriction between ligand unit and linker polymer is shown to be a key parameter. We construct an enhancement diagram that quantifies the multivalent binding affinity compared to monovalent ligands. We conclude that multivalent ligands against influenza viral hemagglutinin ( n = 3), cholera toxin ( n = 5), and anthrax receptor ( n = 7) can outperform monovalent ligands only for a monovalent ligand affinity that exceeds a core-size dependent threshold value. Thus, multivalent drug design needs to balance core size, linker length, as well as monovalent ligand unit affinity.

Usage characteristics and adverse event rates ​of the direct puncture and pull techniques for percutaneous endoscopic gastrostomy in patients with malignant tumors of the upper aerodigestive tract.

BACKGROUND AND STUDY AIMS: Patients with malignant tumors of the upper gastrointestinal tract are at risk of weight loss. Early supportive nutrition therapy is therefore recommended and usually requires placement of a percutaneous endoscopic gastrostomy (PEG). The aim of this study was to compare adverse events and usage characteristics of the direct puncture technique with those of the traditional pull technique when used in patients with endoscopically passable tumors. The primary endpoint was the rate of inflammatory adverse events (AEs) at the gastrostomy fistula. The secondary endpoint was the long-term rate of puncture-site metastases. PATIENTS AND METHODS: One hundred twenty patients (median age 56; IQR 36, 86 years) were randomized and treated per protocol in this prospective open randomized single-center study. Follow-ups were conducted on the third and seventh post-interventional days, after 1, 3 and 6 months and the last follow-up 5 years after intervention. RESULTS: Within the short-term follow-up period of 6 months after PEG placement, AEs were noted in 47 patients (39.2 %). These included 22 inflammations and 16 device dislocations and were mainly found in the puncture group (33 vs. 14 in the pull group) with a significantly increased incidence in the first month after PEG insertion ( P  = 0.001). Evaluation of the 5-year data did not reveal any significant differences. The gastrostomy tube was used in 101 patients (84.2 %) (range 18 days to 5 years). CONCLUSIONS: Our results favor the pull technique for patients with endoscopically passable tumors of the upper gastrointestinal tract due to less short-term adverse events. Both systems contributed equally to secure long-term use.

Spatial Screening of Hemagglutinin on Influenza A Virus Particles: Sialyl-LacNAc Displays on DNA and PEG Scaffolds Reveal the Requirements for Bivalency Enhanced Interactions with Weak Monovalent Binders.

Attachment of the Influenza A virus onto host cells involves multivalent interactions between virus surface hemagglutinin (HA) and sialoside-containing glyco ligands. Despite the development of extremely powerful multivalent binders of the Influenza virus and other viruses, comparably little is known about the optimal spacing of HA ligands, which ought to bridge binding sites within or across the trimeric HA molecules. To explore the criteria for ligand economical high affinity binding, we systematically probed distance-affinity relationships by means of two differently behaving scaffold types based on (i) flexible polyethylene glycol (PEG) conjugates and (ii) rigid self-assembled DNA·PNA complexes. The bivalent scaffolds presented two sialyl-LacNAc ligands in 23-101 Å distance. A combined analysis of binding by means of microscale thermophoresis measurements and statistical mechanics models exposed the inherent limitations of PEG-based spacers. Given the distance requirements of HA, the flexibility of PEG scaffolds is too high to raise the effective concentration of glyco ligands above a value that allows interactions with the low affinity binding site. By contrast, spatial screening with less flexible, self-assembled peptide nucleic acid (PNA)·DNA complexes uncovered a well-defined and, surprisingly, bimodal distance-affinity relationship for interactions of the Influenza A virus HA with bivalent displays of the natural sialyl-LacNAc ligand. Optimal constructs conferred 103-fold binding enhancements with only two ligands. We discuss the existence of secondary binding sites and shine light on the preference for intramolecular rather than intermolecular recognition of HA trimers on the virus surface.

Hydration Effects Turn a Highly Stretched Polymer from an Entropic into an Energetic Spring.

Polyethylene glycol (PEG) is a structurally simple and nontoxic water-soluble polymer that is widely used in medical and pharmaceutical applications as molecular linker and spacer. In such applications, PEG's elastic response against conformational deformations is key to its function. According to text-book knowledge, a polymer reacts to the stretching of its end-to-end separation by a decrease in entropy that is due to the reduction of available conformations, which is why polymers are commonly called entropic springs. By a combination of single-molecule force spectroscopy experiments with molecular dynamics simulations in explicit water, we show that entropic hydration effects almost exactly compensate the chain conformational entropy loss at high stretching. Our simulations reveal that this entropic compensation is due to the stretching-induced release of water molecules that in the relaxed state form double hydrogen bonds with PEG. As a consequence, the stretching response of PEG is predominantly of energetic, not of entropic, origin at high forces and caused by hydration effects, while PEG backbone deformations only play a minor role. These findings demonstrate the importance of hydration for the mechanics of macromolecules and constitute a case example that sheds light on the antagonistic interplay of conformational and hydration degrees of freedom.

Influence of length and flexibility of spacers on the binding affinity of divalent ligands.

We present a quantitative model for the binding of divalent ligand-receptor systems. We study the influence of length and flexibility of the spacers on the overall binding affinity and derive general rules for the optimal ligand design. To this end, we first compare different polymeric models and determine the probability to simultaneously bind to two neighboring receptor binding pockets. In a second step the binding affinity of divalent ligands in terms of the IC50 value is derived. We find that a divalent ligand has the potential to bind more efficiently than its monovalent counterpart only, if the monovalent dissociation constant is lower than a critical value. This critical monovalent dissociation constant depends on the ligand-spacer length and flexibility as well as on the size of the receptor. Regarding the optimal ligand-spacer length and flexibility, we find that the average spacer length should be equal or slightly smaller than the distance between the receptor binding pockets and that the end-to-end spacer length fluctuations should be in the same range as the size of a receptor binding pocket.