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Oxidized model membranes have differential effects on peptide fibril formation, driven by surface attraction, peptide charge and secondary structure stabilization.

MotivationMany membrane peripheral proteins have evolved to transiently interact with the surface of (curved) lipid bilayers. Currently, methods toquantitativelypredict sensing and binding free energies for protein sequences or structures are lacking, and such tools could greatly benefit the discovery of membrane-interacting motifs, as well as theirde novodesign.ResultsHere, we trained a transformer neural network model on molecular dynamics data for>50,000 peptides that is able to accurately predict the (relative) membrane-binding free energy for any given amino acid sequence. Using this information, our physics-informed model is able to classify a peptide’s membrane-associative activity as either non-binding, curvature sensing, or membrane binding. Moreover, this method can be applied to detect membrane-interaction regions in a wide variety of proteins, with comparable predictive performance as state-of-the-art data-driven tools like DREAMM, PPM, and MODA, but with a wider applicability regarding protein diversity, and the added feature to distinguish curvature sensing from general membrane binding.AvailabilityWe made these tools available as a web server, coined Protein-Membrane Interaction predictor (PMIpred), which can be accessed athttps://pmipred.fkt.physik.tu-dortmund.de.

Proteins can specifically bind to curved membranes through curvature-induced hydrophobic lipid packing defects. The chemical diversity among such curvature “sensors” challenges our understanding of how they differ from general membrane “binders” that bind without curvature selectivity. Here, we combine an evolutionary algorithm with coarse-grained molecular dynamics simulations (Evo-MD) to resolve the peptide sequences that optimally recognize the curvature of lipid membranes. We subsequently demonstrate how a synergy between Evo-MD and a neural network (NN) can enhance the identification and discovery of curvature sensing peptides and proteins. To this aim, we benchmark a physics-trained NN model against experimental data and show that we can correctly identify known sensors and binders. We illustrate that sensing and binding are phenomena that lie on the same thermodynamic continuum, with only subtle but explainable differences in membrane binding free energy, consistent with the serendipitous discovery of sensors., Science advances;9(11)

To mount a secondary immune response, dendritic cells must process antigens, migrate to lymph nodes and form synapses with T cells. Critical to 3D migration and mechano-sensing is the nucleus, which is the size-limiting barrier for navigation through gaps in the extracellular matrix. Here, we show that spherical deformations of the dendritic cell nuclear membrane occur following inflammatory activation, as a partial result of reduced cell adhesion. By re-programming the nucleus, dendritic cells are less confined by complex 3D environments and can overcome the typical 2 – 3-micron pore limit for migration. Nuclear deformation is concomitant with the redistribution and bundling of perinuclear actin away from the nucleus, reducing force across Nesprin-2G based linker of nucleoskeleton and cytoskeleton (LINC) complexes. Furthermore, through phosphoproteomics, we connect this nuclear deformation to cofilin-1 serine 41 phosphorylation. This phosphorylation further drives nuclear deformation and bundles cofilin-1 with F-actin into a peri-nuclear halo-shaped micro-domain. In summary, these data describe novel signaling events through which dendritic cells deform their nucleus to enhance their migratory capacity; a molecular event that may be re-capitulated in other contexts such as wound healing and cancer.

The self-assembly of peptides into supramolecular fibril structures has been linked to neurodegenerative diseases such as Alzheimer’s disease but has also been observed in functional roles. Peptides are physiologically exposed to crowded environments of biomacromolecules, and particularly membrane lipids, within a cellular milieu. Previous research has shown that membranes can both accelerate and inhibit peptide self-assembly. Here, we studied the impact of biomimetic membranes that mimic cellular oxidative stress and compared this to mammalian and bacterial membranes. Using molecular dynamics simulations and experiments, we propose a model that explains how changes in peptide-membrane binding, electrostatics, and peptide secondary structure stabilization determine the nature of peptide self-assembly. We explored the influence of zwitterionic (POPC), anionic (POPG) and oxidized (PazePC) phospholipids, as well as cholesterol, and mixtures thereof, on the self-assembly kinetics of the amyloid β (1–40) peptide (Aβ40), linked to Alzheimer’s disease, and the amyloid-forming antimicrobial peptide uperin 3.5 (U3.5). We show that the presence of an oxidized lipid had similar effects on peptide self-assembly as the bacterial mimetic membrane. While Aβ40 fibril formation was accelerated, U3.5 aggregation was inhibited by the same lipids at the same peptide-to-lipid ratio. We attribute these findings and peptide-specific effects to differences in peptide-membrane adsorption with U3.5 being more strongly bound to the membrane surface and stabilized in an α-helical conformation compared to Aβ40. Different peptide-to-lipid ratios resulted in different effects. Molecular dynamics simulations provided detailed mechanistic insights into the peptide-lipid interactions and secondary structure stability. We found that electrostatic interactions are a primary driving force for peptide-membrane interaction, enabling us to propose a model for predictions how cellular changes might impact peptide self-assembly in vivo, and potentially impact related diseases.

Membrane curvature plays an essential role in the organization and trafficking of membrane associated proteins. Comparison or prediction of the experimentally resolved protein concentrations adopted at different membrane curvatures requires direct quantification of the relative partitioning free energy. Here, we present a highly efficient and simple to implement a free-energy calculation method which is able to directly resolve the relative partitioning free energy of proteins as a direct function of membrane curvature, i.e., a curvature sensing profile, within (coarse-grained) molecular dynamics simulations. We demonstrate its utility by resolving these profiles for two known curvature sensing peptides, namely ALPS and α-synuclein, for a membrane curvature ranging from -1/6.5 to +1/6.5 nm-1. We illustrate that the difference in relative partitioning (binding) free energy between these two extrema is only about 13 kBT for both peptides, illustrating that the driving force of curvature sensing is subtle. Furthermore, we illustrate that ALPS and α-synuclein sense curvature via a contrasting mechanism, which is differentially affected by membrane composition. In addition, we demonstrate that the intrinsic spontaneous curvature of both of these peptides lies beyond the range of membrane curvature accessible in micropipette aspiration experiments, being about 1/7 nm -1. Our approach offers an efficient and simple to implement in silico tool for exploring and screening the membrane curvature sensing mechanisms of proteins.

The mitochondrial inner membrane is an integral part of the cellular lipid biosynthesis network. Intramitochondrial lipid transfer shuttles specific lipid species between the two mitochondrial membranes. This pathway is facilitated by designated protein complexes in the intermembrane space. A hetero-dimeric complex of Ups1 and Mdm35 has been identified in yeast to transfer phosphatidic acid from the outer to the inner membrane. Here, we define pH and lipid charge dependency of Ups1 membrane interaction. We show that Ups1 interacts with membranes in a membrane curvature dependent manner. Ups1 predominantly binds to membrane domains of positive curvature. Desorption of phosphatidic acid from positively curved membranes is energetically favorable over flat or negatively curved membranes. Hence, our results demonstrate that Ups1 specifically binds to membrane regions where extraction and insertion of lipids is enhanced.

In biological systems, proteins can be attracted to curved or stretched regions of lipid bilayers by sensing hydrophobic defects in the lipid packing on the membrane surface. Here, we present an efficient end-state free energy calculation method to quantify such sensing in molecular dynamics simulations. We illustrate that lipid packing defect sensing can be defined as the difference in mechanical work required to stretch a membrane with and without a peptide bound to the surface. We also demonstrate that a peptide’s ability to concurrently induce excess leaflet area (tension) and elastic softening – a property we call the ‘characteristic area of sensing’ (CHAOS) – and lipid packing sensing behavior are in fact two sides of the same coin. In essence, defect sensing displays a peptide’s propensity to generate tension. The here-proposed mechanical pathway is equally accurate yet, computationally, about 40 times less costly than the commonly used alchemical pathway (thermodynamic integration), allowing for more feasible free energy calculations in atomistic simulations. This enabled us to directly compare the Martini 2 and 3 coarse-grained and the CHARMM36 atomistic force-fields in terms of relative binding free energies for six representative peptides including the curvature sensor ALPS and two antiviral amphipathic helices (AH). We observed that Martini 3 qualitatively reproduces experimental trends, whilst producing substantially lower (relative) binding free energies and shallower membrane insertion depths compared to atomistic simulations. In contrast, Martini 2 tends to overestimate (relative) binding free energies. Finally, we offer a glimpse into how our end-state based free energy method can enable the inverse design of optimal lipid packing defect sensing peptides when used in conjunction with our recently developed Evolutionary Molecular Dynamics (Evo-MD) method. We argue that these optimized defect sensors – aside from their biomedical and biophysical relevance – can provide valuable targets for the development of lipid force-fields.TOC Graphic

Thermodynamic integration is one of the most established methods to quantify excess free energies between different metastable states. Excess intermolecular interactions in surfactant assemblies are on the scale of the energy of thermal fluctuations. Therefore, these materials can be deformed and topologically altered via relatively small mechanical stresses. It is thus intuitive to design reaction paths and associated order parameters that exploit the "soft" nature of these materials to mechanically rather than alchemically morph surfactant assemblies from state to state. Here, we propose a novel method coined "density field thermodynamic integration" (DFTI) that adopts the universality and transferability of alchemical methods while simultaneously exploiting the soft excess interactions between surfactant molecules. DFTI was designed for a rapid quantification of the free energy differences between different metastable structures in soft fluid materials. The DFTI method uses an external field coupled to the local density to mechanically morph the system between metastable states of interest. Here, we explored the capability of the DFTI method to swiftly and accurately calculate free energy differences between states. To this aim, we studied two different coarse-grained lipidic surfactant systems: (i) a fusion stalk and (ii) a worm-like micelle. Our results illustrate that DFTI can provide an efficient, versatile, and rather reliable method to calculate the free energy differences between surfactant assemblies.

Physiological membrane vesicles are built to separate reaction spaces in a stable manner, even when they accidentally collide or are kept in apposition by spatial constraints in the cell. This requires a natural resistance to fusion and mixing of their content, which originates from substantial energetic barriers to membrane fusion [1]. To facilitate intracellular membrane fusion reactions in a controlled manner, proteinaceous fusion machineries have evolved. An important open question is whether protein fusion machineries actively pull the fusion reaction over the present free energy barriers, or whether they rather catalyze fusion by lowering those barriers. At first sight, fusion proteins such as SNARE complexes and viral fusion proteins appear to act as nano-machines, which mechanically transduce force to the membranes and thereby overcome the free energy barriers [2,3]. Whether fusion proteins additionally alter the free energy landscape of the fusion reaction via catalytic roles is less obvious. This is a question that we shall discuss in this review, with particular focus on the influence of the eukaryotic SNARE-dependent fusion machinery on the final step of the reaction, the formation and expansion of the fusion pore.

The aggregation of peptides into amyloid fibrils has been linked to ageing-related diseases, such as Alzheimer's and type 2 diabetes. Interfaces, particularly those with large nanostructured surfaces, can affect the kinetics of peptide aggregation, which ranges from complete inhibition to strong acceleration. While a number of physiochemical parameters determine interfacial effects, we focus here on the role of nanoparticle (NP) size and curvature. We used thioflavin T (ThT) fluorescence assays to demonstrate the size-dependent effects of NPs on amyloid fibril formation for the peptides Aβ

The islet amyloid polypeptide (IAPP) is a regulatory peptide that can aggregate into fibrillar structures associated with type 2 diabetes. In this study, the IAPP21-27 segment was modified with a biotin linker at the N-terminus (Btn-GNNFGAIL) to immobilize peptide fibrils on streptavidin-coated surfaces. Key residues for fibril formation of the N-terminal biotinylated IAPP21-27 segment were identified by using an alanine scanning approach combined with molecular dynamics simulations, thioflavin T fluorescence measurements, and scanning electron microscopy. Significant contributions of phenylalanine (F23), leucine (L27), and isoleucine (I26) for the fibrillation of the short peptide segment were identified. The fibril morphologies of the peptide variants differed depending on their primary sequence, ranging from flexible and semiflexible to stiff and crystal-like structures. These insights could advance the design of new functional hybrid bionanomaterials and fibril-engineered surface coatings using short peptide segments. To validate this concept, the biotinylated fibrils were immobilized on streptavidin-coated surfaces under spatial control.

The twin-arginine translocation (Tat) system serves to translocate folded proteins across energy-transducing membranes in bacteria, archaea, plastids, and some mitochondria. In Escherichia coli, TatA, TatB, and TatC constitute functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) followed by an amphipathic helix. The TMHs of TatA and TatB generate a hydrophobic mismatch with the membrane, as the helices comprise only 12 consecutive hydrophobic residues; however, the purpose of this mismatch is unclear. Here, we shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both and analyzed effects on translocon function and assembly. We found the WT length helices functioned best, but some variation was clearly tolerated. Defects in function were exacerbated by simultaneous mutations in TatA and TatB, indicating partial compensation of mutations in each by the other. Furthermore, length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residue-length is important but not essential for this interaction and translocon assembly. To also address potential effects of helix length on TatA interactions, we characterized these interactions by molecular dynamics simulations, after having characterized the TatA assemblies by metal-tagging transmission electron microscopy. In these simulations, we found that interacting short TMHs of larger TatA assemblies were thinning the membrane and-together with laterally-aligned tilted amphipathic helices-generated a deep V-shaped membrane groove. We propose the 12 consecutive hydrophobic residues may thus serve to destabilize the membrane during Tat transport, and their conservation could represent a delicate compromise between functionality and minimization of proton leakage.

The occurrence of linear cholesterol-recognition motifs in alpha-helical transmembrane domains has long been debated. Here, we demonstrate the ability of a genetic algorithm guided by coarse-grained molecular dynamics simulations—a method coined evolutionary molecular dynamics (Evo-MD)—to directly resolve the sequence which maximally attracts cholesterol for single-pass alpha-helical transmembrane domains (TMDs). We illustrate that the evolutionary landscape of cholesterol attraction in membrane proteins is characterized by a sharp, well-defined global optimum. Surprisingly, this optimal solution features an unusual short hydrophobic block, consisting of typically only eight small hydrophobic amino acids, surrounded by three successive lysines. Owing to the membrane thickening effect of cholesterol, cholesterol-enriched ordered phases favor TMDs characterized by a long rather than a too short hydrophobic length (a negative hydrophobic mismatch). However, this short hydrophobic pattern evidently offers a pronounced net advantage for the attraction of free cholesterol in both coarse-grained and atomistic simulations. We illustrate that optimal cholesterol attraction is in fact based on the superposition of two distinct thermodynamic attraction mechanisms. In addition, we explore the evolutionary occurrence and feasibility of these two mechanisms by analyzing existing databases of membrane proteins and through the direct expression of analogous short hydrophobic sequences in live cell assays. The puzzling sequence variability of proposed linear cholesterol-recognition motifs is indicative of a sub-optimal membrane-mediated attraction of cholesterol which markedly differs from ligand binding based on shape compatibility.Significance StatementOur work demonstrates how a synergy between evolutionary algorithms and high-throughput coarse-grained molecular dynamics can yield fundamentally new insights into the evolutionary fingerprints of protein-mediated lipid sorting. We illustrate that the evolutionary landscape of cholesterol attraction in isolated transmembrane domains is characterized by a well-defined global optimum. In contrast, sub-optimal attraction of cholesterol is associated with a diverse solution space and features a high sequence variability despite acting on the same unique molecule. The contrasting physicochemical nature of the resolved attraction optimum suggests that cholesterol attraction via linear motifs does not pose a dominant pressure on the evolution of transmembrane proteins.

The aggregation of peptides into amyloid fibrils is linked to ageing-related diseases, such as Alzheimer’s disease and type 2 diabetes. Interfaces, particularly those with large nanostructured surface areas, can affect the kinetics of peptide aggregation, ranging from a complete inhibition to strong acceleration. While a number of physiochemical parameters determine interface effects, we here focus on the role of nanoparticle curvature for the aggregation of the amyloidogenic peptides Aβ40, NNFGAIL, GNNQQNY and VQIYVK. Nanoparticles (NPs) provided a surface for peptide monomers to adsorb, enabling the nucleation into oligomers and fibril formation. High surface curvature, however, destabilized prefibrillar structures, providing an explanation for inhibitory effects on fibril growth. Thioflavin T (ThT) fluorescence assays as well as dynamic light scattering (DLS), atomic force microscopy (AFM) and electron microscopy experiments revealed NP size-dependent effects on amyloid fibril formation, with differences between the peptides. While 5 nm gold NPs (AuNP-5) retarded or inhibited the aggregation of most peptides, larger 20 nm gold NPs (AuNP-20) tended to accelerate peptide aggregation. Molecular dynamics (MD) studies demonstrated that NPs’ ability to catalyze or inhibit oligomer formation was influenced by the oligomer stability at curved interfaces which was lower at more highly curved surfaces. Differences in the NP effects for the peptides resulted from the peptide properties (size, aggregation propensity) and concomitant surface binding affinities. The results can be applied to the design of future nanostructured materials for defined applications.

The Tat system has the unique purpose to translocate folded proteins across energy-transducing membranes. It occurs in bacteria and archaea, as well as in eukaryotic organelles of bacterial origin. In the bacterial model system Escherichia coli, the three components TatA, TatB, and TatC assemble to functional translocons. TatA and TatB both possess an N-terminal transmembrane helix (TMH) that is followed by an amphipathic helix (APH). The TMHs of TatA and TatB generate a hydrophobic mismatch with only 12 consecutive hydrophobic residues that span the membrane. We shortened or extended this stretch of hydrophobic residues in either TatA, TatB, or both, and analyzed effects on transport functionality and translocon assembly. The wild type length functioned best but was not an absolute requirement, as some variation was clearly tolerated. Defects of shortenings or extensions were enhanced by simultaneous mutations in TatA and TatB, indicating partial compensations of mutations in TatA by wild type TatB or vice versa. Length variation in TatB destabilized TatBC-containing complexes, revealing that the 12-residues-length is important for Tat component interactions and translocon assembly. To also address potential effects on TatA associations, we characterized these by metal tagging transmission electron microscopy and carried out molecular dynamics simulations. In these simulations, interacting short TMHs of larger TatA assemblies were thinning the membrane together with laterally aligned tilted APHs that generated a deep V-shaped groove. The conserved length of 12 hydrophobic residues may thus not only be important for translocon interactions, but also for a membrane destabilization during Tat transport. If this is the case, the specific short length could be a compromise between functionality and proton leakage minimization.

Heterogeneities (e.g., membrane proteins and lipid domains) and deformations (e.g., highly curved membrane regions) in biological lipid membranes cause lipid packing defects that may trigger functional sorting of lipids and membrane-associated proteins. To study these phenomena in a controlled and efficient way within molecular simulations, we developed an external field protocol that artificially enhances packing defects in lipid membranes by enforcing local thinning of a flat membrane region. For varying lipid compositions, we observed strong thinning-induced depletion or enrichment, depending on the lipid's intrinsic shape and its effect on a membrane's elastic modulus. In particular, polyunsaturated and lysolipids are strongly attracted to regions high in packing defects, whereas phosphatidylethanolamine (PE) lipids and cholesterol are strongly repelled from it. Our results indicate that externally imposed changes in membrane thickness, area, and curvature are underpinned by shared membrane elastic principles. The observed sorting toward the thinner membrane region is in line with the sorting expected for a positively curved membrane region. Furthermore, we have demonstrated that the amphipathic lipid packing sensor (ALPS) protein motif, a known curvature and packing defect sensor, is effectively attracted to thinner membrane regions. By extracting the force that drives amphipathic molecules toward the thinner region, our thinning protocol can directly quantify and score the lipid packing sensing of different amphipathic molecules. In this way, our protocol paves the way toward high-throughput exploration of potential defect- and curvature-sensing motifs, making it a valuable addition to the molecular simulation toolbox.

Biological transmission of vesicular content occurs by opening of a fusion pore. Recent experimental observations have illustrated that fusion pores between vesicles that are docked by an extended flat contact zone are located at the edge (vertex) of this zone. We modeled this experimentally observed scenario by coarse-grained molecular simulations and elastic theory. This revealed that fusion pores experience a direct attraction toward the vertex. The size adopted by the resulting vertex pore strongly depends on the apparent contact angle between the adhered vesicles even in the absence of membrane surface tension. Larger contact angles substantially increase the equilibrium size of the vertex pore. Because the cellular membrane fusion machinery actively docks membranes, it facilitates a collective expansion of the contact zone and increases the contact angle. In this way, the fusion machinery can drive expansion of the fusion pore by free energy equivalents of multiple tens of k B T from a distance and not only through the fusion proteins that reside within the fusion pore.

The islet amyloid polypeptide (IAPP) is a regulatory peptide that can aggregate into fibrillar structures associated with type 2 diabetes. In this study, the IAPP

peerReviewed

Society is increasingly exposed to nanoparticles as they are ubiquitous in nature and introduced as man-made air pollutants and as functional ingredients in cosmetic products as well as in nanomedicine. Nanoparticles differ in size, shape and material properties. In addition to their intended function, the side effects on biochemical processes in organisms remain unclear. Nanoparticles can significantly influence the nucleation and aggregation process of peptides. The development of several neurodegenerative diseases, such as Alzheimer's disease, is related to the aggregation of peptides into amyloid fibrils. However, there is no comprehensive or universal mechanism to predict or explain apparent acceleration or inhibition of these aggregation processes. In this work, selected studies and possible mechanisms for amyloid peptide nucleation and aggregation, in the presence of nanoparticles, are highlighted. These studies are discussed in the context of recent data from our group on the role of gold nanoparticles in amyloid peptide aggregation using experimental methods and large-scale molecular dynamics simulations. A complex interplay of the surface properties of the nanoparticles, the properties of the peptides, as well as the resulting forces between both the nanoparticles and the peptides, appear to determine whether amyloid peptide aggregation is influenced, catalysed or inhibited by the presence of nanoparticles.

Membrane fusion in eukaryotic cells mediates the biogenesis of organelles, vesicular traffic between them, and exo- and endocytosis of important signalling molecules, such as hormones and neurotransmitters. Distinct tasks in intracellular membrane fusion have been assigned to conserved protein systems. Tethering proteins mediate the initial recognition and attachment of membranes, whereas SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes are considered as the core fusion engine. SNARE complexes provide mechanical energy to distort membranes and drive them through a hemifusion intermediate towards the formation of a fusion pore. This last step is highly energy-demanding. Here we combine the in vivo and in vitro fusion of yeast vacuoles with molecular simulations to show that tethering proteins are critical for overcoming the final energy barrier to fusion pore formation. SNAREs alone drive vacuoles only into the hemifused state. Tethering proteins greatly increase the volume of SNARE complexes and deform the site of hemifusion, which lowers the energy barrier for pore opening and provides the driving force. Thereby, tethering proteins assume a crucial mechanical role in the terminal stage of membrane fusion that is likely to be conserved at multiple steps of vesicular traffic. We therefore propose that SNAREs and tethering proteins should be considered as a single, non-dissociable device that drives fusion. The core fusion machinery may then be larger and more complex than previously thought.

Biological membrane fusion proceeds via an essential topological transition of the two membranes involved. Known players such as certain lipid species and fusion proteins are generally believed to alter the free energy and thus the rate of the fusion reaction. Quantifying these effects by theory poses a major challenge since the essential reaction intermediates are collective, diffusive and of a molecular length scale. We conducted molecular dynamics simulations in conjunction with a state-of-the-art string method to resolve the minimum free-energy path of the first fusion intermediate state, the so-called stalk. We demonstrate that the isolated transmembrane domains (TMDs) of fusion proteins such as SNARE molecules drastically lower the free energy of both the stalk barrier and metastable stalk, which is not trivially explained by molecular shape arguments. We relate this effect to the local thinning of the membrane (negative hydrophobic mismatch) imposed by the TMDs which favors the nearby presence of the highly bent stalk structure or prestalk dimple. The distance between the membranes is the most crucial determinant of the free energy of the stalk, whereas the free-energy barrier changes only slightly. Surprisingly, fusion enhancing lipids, i.e., lipids with a negative spontaneous curvature, such as PE lipids have little effect on the free energy of the stalk barrier, likely because of its single molecular nature. In contrast, the lipid shape plays a crucial role in overcoming the hydration repulsion between two membranes and thus rather lowers the total work required to form a stalk.

The question of how amyloid fibril formation is influenced by surfaces is crucial for a detailed understanding of the process in vivo. We applied a combination of kinetic experiments and molecular dynamics simulations to elucidate how (model) surfaces influence fibril formation of the amyloid-forming sequences of prion protein SUP35 and human islet amyloid polypeptide. The kinetic data suggest that structural reorganization of the initial peptide corona around colloidal gold nanoparticles is the rate-limiting step. The molecular dynamics simulations reveal that partial physisorption to the surface results in the formation of aligned monolayers, which stimulate the formation of parallel, critical oligomers. The general mechanism implies that the competition between the underlying peptide–peptide and peptide–surface interactions must strike a balance to accelerate fibril formation. peerReviewed

Die Frage, wie Amyloidfibrillenbildung von Oberflachen beeinflusst wird, ist entscheidend fur das Verstandnis des Prozesses in vivo. Wir haben eine Kombination von kinetischen Experimenten und Molekuldynamiksimulationen angewendet, um aufzuklaren, wie (Modell-)Oberflachen die Fibrillenbildung der amyloidbildenden Sequenzen des Prionenproteins SUP35 und des Insel-Amyloid-Polypeptids beeinflussen. Kinetische Daten legen nahe, dass eine Restrukturierung der initialen Peptidkorona um die kolloidalen Goldnanopartikel der zeitlimitierende Schritt ist. Molekuldynamiksimulationen zeigen, dass partielle Physisorption an der Oberflache zur Bildung von geordneten Monolagen fuhrt, die die Bildung von parallelen, kritischen Oligomeren stimuliert. Der generelle Mechanismus beinhaltet, dass die zugrundeliegende Peptid-Peptid- und die Peptid-Oberflachen-Wechselwirkung in der gleichen Grosenordnung liegen mussen, um die Fibrillenbildung zu beschleunigen.

Investigating the adsorption of peptides on inorganic surfaces, on the molecular level, is fundamental for medicinal and analytical applications. Peptides can be potent as linkers between surfaces and living cells in biochips or in implantation medicine. Here, we studied the adsorption process of the positively charged pentapeptide RTHRK, a recently identified binding sequence for surface oxidized silicon, and novel analogues thereof to negatively charged mica surfaces. Homogeneous formation of monolayers in the nano- and low micromolar peptide concentration range was observed. We propose an alternative and efficient method to both quantify binding affinity and follow adhesion behavior. This method makes use of the thermodynamic relationship between surface coverage, measured by atomic force microscopy (AFM), and the concomitant free energy of adhesion. A knowledge-based fit to the autocorrelation of the AFM images was used to correct for a biased surface coverage introduced by the finite lateral resolution of the AFM. Binding affinities and mechanisms were further explored by large scale molecular dynamics (MD) simulations. The combination of well validated MD simulations with topological data from AFM revealed a better understanding of peptide adsorption processes on the atomistic scale. We demonstrate that binding affinity is strongly determined by a peptide's ability to form salt bridges and hydrogen bonds with the surface lattice. Consequently, differences in hydrogen bond formation lead to substantial differences in binding affinity despite conservation of the peptide's overall charge. Further, MD simulations give access to relative changes in binding energy of peptide variations in comparison to a lead compound.

Constitutive membrane fusion within eukaryotic cells is thought to be controlled at its initial steps, membrane tethering and SNARE complex assembly, and to rapidly proceed from there to full fusion. Although theory predicts that fusion pore expansion faces a major energy barrier and might hence be a rate‐limiting and regulated step, corresponding states with non‐expanding pores are difficult to assay and have remained elusive. Here, we show that vacuoles in living yeast are connected by a metastable, non‐expanding, nanoscopic fusion pore. This is their default state, from which full fusion is regulated. Molecular dynamics simulations suggest that SNAREs and the SM protein‐containing HOPS complex stabilize this pore against re‐closure. Expansion of the nanoscopic pore to full fusion can thus be triggered by osmotic pressure gradients, providing a simple mechanism to rapidly adapt organelle volume to increases in its content. Metastable, nanoscopic fusion pores are then not only a transient intermediate but can be a long‐lived, physiologically relevant and regulated state of SNARE‐dependent membrane fusion.

The productive fusion pore in membrane fusion is generally thought to be toroidally shaped. Theoretical studies and recent experiments suggest that its formation, in some scenarios, may be preceded by an initial pore formed near the rim of the extended hemifusion diaphragm (HD), a rim-pore. This rim-pore is characterized by a nontoroidal shape that changes with size. To determine this shape as well as the free energy along the pathway of rim-pore expansion, we derived a simple analytical free energy model. We argue that dilation of HD material via expansion of a rim-pore is favored over a regular, circular pore. Further, the expanding rim-pore faces a free energy barrier that linearly increases with HD size. In contrast, the tension required to expand the rim-pore decreases with HD size. Pore flickering, followed by sudden opening, occurs when the tension in the HD competes with the line energy of the rim-pore, and the rim-pore reaches its equilibrium size before reaching the critical pore size. The experimental observation of flickering and closing fusion pores (kiss-and-run) is very well explained by the observed behavior of rim-pores. Finally, the free energy landscape of rim-pore expansion/HD dilation may very well explain why some cellular fusion reactions, in their attempt to minimize energetic costs, progress via alternative formation and dilation of microscopic hemifusion intermediates.

Coarse-grained molecular dynamics simulations are applied to explore the experimentally observed ability of the liquid-ordered (lo)/liquid-disordered (ld) phase boundary to facilitate viral membrane fusion. Surprisingly, a formed fusion stalk can be both attracted (i.e., stalkophilic) and repelled (i.e., stalkophobic) by the lo/ld phase boundary. The phase boundary becomes stalkophilic if the lo phase constituents have the larger negative spontaneous curvature. In such a case, location of the highly curved stalk near the less-ordered and thus (relatively) softer boundary region becomes energetically favorable. These insights may explain why modeled viral fusion occurs preferentially near the lo/ld phase boundary.

A complete physical description of membrane remodeling processes, such as fusion or fission, requires knowledge of the underlying free energy landscapes, particularly in barrier regions involving collective shape changes, topological transitions, and high curvature, where Canham-Helfrich (CH) continuum descriptions may fail. To calculate these free energies using atomistic simulations, one must address not only the sampling problem due to high free energy barriers, but also an orthogonal sampling problem of combinatorial complexity stemming from the permutation symmetry of identical lipids. Here, we solve the combinatorial problem with a permutation reduction scheme to map a structural ensemble into a compact, nondegenerate subregion of configuration space, thereby permitting straightforward free energy calculations via umbrella sampling. We applied this approach, using a coarse-grained lipid model, to test the CH description of bending and found sharp increases in the bending modulus for curvature radii below 10 nm. These deviations suggest that an anharmonic bending term may be required for CH models to give quantitative energetics of highly curved states.

SNAREs fuse membranes in several steps. Trans-SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C-termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C-termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C-termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C-termini permits SNARE activation, trans-SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans-SNARE complex carries a protein tag. Thus, both sides of a trans-SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C-termini cooperate in opening the fusion pore by locally deforming the inner leaflets.

Electrophoresis is widely used to determine the electrostatic potential of colloidal particles. Oil droplets in pure water show negative or positive electrophoretic mobilities depending on the pH. This is commonly attributed to the adsorption of hydroxyl or hydronium ions, resulting in a negative or positive surface charge, respectively. This explanation, however, is not in agreement with the difference in isoelectric point and point of zero charge observed in experiment. Here we present molecular dynamics simulations of oil droplets in water in the presence of an external electric field but in the absence of any ions. The simulations reproduce the negative sign and the order of magnitude of the oil droplet mobilities at the point of zero charge in experiment. The electrostatic potential in the oil with respect to the water phase, induced by anisotropic dipole orientation in the interface, is positive. Our results suggest that electrophoretic mobility does not always reflect the net charge or electrostatic potential of a suspended liquid droplet and, thus, the interpretation of electrophoresis in terms of purely continuum effects may need to be reevaluated. (C) 2007 Elsevier Inc. All rights reserved.

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes., Clustering of proteins in the plasma membrane plays an important role in the regulation of both cellular signalling and membrane remodelling. Milovanovic et al. demonstrate that mismatch between transmembrane domain length and the lipid bilayer thickness is sufficient to drive clustering of SNARE proteins.

Over the past 20 years, it has been widely accepted that membrane fusion proceeds via a hemifusion step before opening of the productive fusion pore. An initial hourglass-shaped lipid structure, the fusion stalk, is formed between the adjacent membrane leaflets (cis leaflets). It remains controversial if and how fusion proteins drive the subsequent transition (expansion) of the stalk into a fusion pore. Here, we propose a comprehensive and consistent thermodynamic understanding in terms of the underlying free-energy landscape of stalk expansion. We illustrate how the underlying free energy landscape of stalk expansion and the concomitant pathway is altered by subtle differences in membrane environment, such as leaflet composition, asymmetry, and flexibility. Nonleaky stalk expansion (stalk widening) requires the formation of a critical trans-leaflet contact. The fusion machinery can mechanically enforce trans-leaflet contact formation either by directly enforcing the trans-leaflets in close proximity, or by (electrostatically) condensing the area of the cis leaflets. The rate of these fast fusion reactions may not be primarily limited by the energetics but by the forces that the fusion proteins are able to exert.

Summary The influenza virus is surrounded by an envelope composed of a lipid bilayer and integral membrane proteins. Understanding the structural dynamics of the membrane envelope provides biophysical insights into aspects of viral function, such as the wide-ranging survival times of the virion in different environments. We have combined experimental data from X-ray crystallography, nuclear magnetic resonance spectroscopy, cryo-electron microscopy, and lipidomics to build a model of the intact influenza A virion. This is the basis of microsecond-scale coarse-grained molecular dynamics simulations of the virion, providing simulations at different temperatures and with varying lipid compositions. The presence of the Forssman glycolipid alters a number of biophysical properties of the virion, resulting in reduced mobility of bilayer lipid and protein species. Reduced mobility in the virion membrane may confer physical robustness to changes in environmental conditions. Our simulations indicate that viral spike proteins do not aggregate and thus are competent for multivalent immunoglobulin G interactions., Graphical Abstract, Highlights • Microsecond timescale molecular dynamics simulations of enveloped influenza virion • Forssman glycolipid alters the biophysical properties of the influenza A virion • Spacing of the spike glycoproteins is compatible with bivalent antibody association • Mobility of viral proteins influences distribution of lipids in the viral envelope, Reddy et al. performed the first microsecond timescale coarse-grained molecular dynamics simulations of enveloped virions in explicit solvent. The simulated properties of the influenza A virion were consistent with experimental measurements, and revealed that the Forssman glycolipid affects several biophysical characteristics of the virion.

The captions of supporting information files Figures S1-S4 were erroneously omitted. The supporting information captions read

SNARE molecules are the core constituents of the protein machinery that facilitate fusion of synaptic vesicles with the presynaptic plasma membrane, resulting in the release of neurotransmitter. On a molecular level, SNARE complexes seem to play a quite versatile and involved role during all stages of fusion. In addition to merely triggering fusion by forcing the opposing membranes into close proximity, SNARE complexes are now seen to also overcome subsequent fusion barriers and to actively guide the fusion reaction up to the expansion of the fusion pore. Here, we review recent advances in the understanding of SNARE-mediated membrane fusion by molecular simulations.

Neurotransmitter release at the synapse requires fusion of synaptic vesicles with the presynaptic plasma membrane. SNAREs are the core constituents of the protein machinery responsible for this membrane fusion, but the actual fusion mechanism remains unclear. Here, we have simulated neuronal SNARE-mediated membrane fusion in molecular detail. In our simulations, membrane fusion progresses through an inverted micelle fusion intermediate before reaching the hemifused state. We show that at least one single SNARE complex is required for fusion, as has also been confirmed in a recent in vitro single-molecule fluoresence study. Further, the transmembrane regions of the SNAREs were found to play a vital role in the initiation of fusion by causing distortions of the lipid packing of the outer membrane leaflets, and the C termini of the transmembrane regions are associated with the formation of the fusion pores. The inherent mechanical stress in the linker region of the SNARE complex was found to drive both the subsequent formation and expansion of fusion pores. Our simulations also revealed that the presence of homodimerizations between the transmembrane regions leads to the formation of unstable fusion intermediates that are under high curvature stress. We show that multiple SNARE complexes mediate membrane fusion in a cooperative and synchronized process. Finally, we show that after fusion, the zipping of the SNAREs extends into the membrane region, in agreement with the recently resolved X-ray structure of the fully assembled state.