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Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and stoichiometric loading of vesicles with single-molecule control., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Although cell membranes exist in excess of water under physiological conditions, there are a number of biochemical processes, such as adsorption of biomacromolecules or membrane fusion events, that require partial or even complete transient dehydration of lipid membranes. Even though the dehydration process is crucial for understanding all fusion events, still little is known about the structural adaptation of lipid membranes when their interfacial hydration layer is perturbed. Here, we present the study of the nanoscale structural reorganization of phase-separated, supported lipid bilayers (SLBs) under a wide range of hydration conditions. Model lipid membranes were characterised using a combination of fluorescence microscopy and atomic force microscopy and, crucially, without applying any chemical or physical modifications that have previously been considered essential for maintaining the membrane integrity upon dehydration. We revealed that decreasing the hydration state of the membrane leads to an enhanced mixing of lipids characteristic of the liquid-disordered (L d ) phase with those forming the liquid-ordered (L o ) phase. This is associated with a 2-fold decrease in the hydrophobic mismatch between the L d and L o lipid phases and a 3-fold decrease in the line tension for the fully desiccated membrane. Importantly, the observed changes in the hydrophobic mismatch, line tension, and lipid miscibility are fully reversible upon subsequent rehydration of the membrane. These findings provide a deeper insight into the fundamental processes, such as cell-cell fusion, that require partial dehydration at the interface of two membranes.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Particle size is an important characteristic of materials with a direct effect on their physicochemical features. Besides nanoparticles, particle size and surface curvature are particularly important in the world of lipids and cellular membranes as the cell membrane undergoes conformational changes in many biological processes which leads to diverging local curvature values. On account of that, it is important to develop cost-effective, rapid and sufficiently precise systems that can measure the surface curvature on the nanoscale that can be translated to size for spherical particles. As an alternative approach for particle characterization, we present flexible DNA nanodevices that can adapt to the curvature of the structure they are bound to. The curvature sensors use Fluorescence Resonance Energy Transfer (FRET) as the transduction mechanism on the single-molecule level. The curvature sensors consist of segmented DNA origami structures connected via flexible DNA linkers incorporating a FRET pair. The activity of the sensors was first demonstrated with defined binding to different DNA origami geometries used as templates. Then the DNA origami curvature sensors were applied to measure spherical silica beads having different size, and subsequently on lipid vesicles. With the designed sensors, we could reliably distinguish different sized nanoparticles within a size range of 50-300 nm as well as the bending angle range of 50-180°. This study helps with the development of more advanced modular-curvature sensing devices that are capable of determining the sizes of nanoparticles and biological complexes.

DNA origami is an extremely versatile nanoengineering tool with widespread applicability in various fields of research, including membrane physiology and biophysics. The possibility to easily modify DNA strands with lipophilic moieties enabled the recent development of a variety of membrane-active DNA origami devices. Biological membranes, as the core barriers of the cells, display vital structural and functional roles. Therefore, lipid bilayers are widely popular targets of DNA origami nanotechnology for synthetic biology and biomedical applications. In this chapter, we summarize the typical experimental methods used to investigate the interaction of DNA origami with synthetic membrane models. Herein, we present detailed protocols for the production of lipid model membranes and characterization of membrane-targeted DNA origami nanostructures using different microscopy approaches., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Upscaling motor protein activity to perform work in man-made devices has long been an ambitious goal in bionanotechnology. The use of hierarchical motor assemblies, as realized in sarcomeres, has so far been complicated by the challenges of arranging sufficiently high numbers of motor proteins with nanoscopic precision. Here, we describe an alternative approach based on actomyosin cortex-like force production, allowing low complexity motor arrangements in a contractile meshwork that can be coated onto soft objects and locally activated by ATP. The design is reminiscent of a motorized exoskeleton actuating protein-based robotic structures from the outside. It readily supports the connection and assembly of micro-three-dimensional printed modules into larger structures, thereby scaling up mechanical work. We provide an analytical model of force production in these systems and demonstrate the design flexibility by three-dimensional printed units performing complex mechanical tasks, such as microhands and microarms that can grasp and wave following light activation., (© 2022. The Author(s).)

Signal transmission in neurons goes along with changes in the transmembrane potential. To report them, different approaches, including optical voltage-sensing dyes and genetically encoded voltage indicators, have evolved. Here, we present a DNA nanotechnology-based system and demonstrated its functionality on liposomes. Using DNA origami, we incorporated and optimized different properties such as membrane targeting and voltage sensing modularly. As a sensing unit, we used a hydrophobic red dye anchored to the membrane and an anionic green dye at the DNA to connect the nanostructure and the membrane dye anchor. Voltage-induced displacement of the anionic donor unit was read out by fluorescence resonance energy transfer (FRET) changes of single sensors attached to liposomes. A FRET change of ∼5% for ΔΨ = 100 mV was observed. The working mechanism of the sensor was rationalized by molecular dynamics simulations. Our approach holds potential for an application as nongenetically encoded membrane sensors.

Self-assembly of biomembranes results from the intricate interactions between water and the lipids' hydrophilic head groups. Therefore, the lipid-water interplay strongly contributes to modulating membrane architecture, lipid diffusion, and chemical activity. Here, we introduce a new method of obtaining dehydrated, phase-separated, supported lipid bilayers (SLBs) solely by controlling the decrease of their environment's relative humidity. This facilitates the study of the structure and dynamics of SLBs over a wide range of hydration states. We show that the lipid domain structure of phase-separated SLBs is largely insensitive to the presence of the hydration layer. In stark contrast, lipid mobility is drastically affected by dehydration, showing a 6-fold decrease in lateral diffusion. At the same time, the diffusion activation energy increases approximately 2-fold for the dehydrated membrane. The obtained results, correlated with the hydration structure of a lipid molecule, revealed that about six to seven water molecules directly hydrating the phosphocholine moiety play a pivotal role in modulating lipid diffusion. These findings could provide deeper insights into the fundamental reactions where local dehydration occurs, for instance during cell-cell fusion, and help us better understand the survivability of anhydrobiotic organisms. Finally, the strong dependence of lipid mobility on the number of hydrating water molecules opens up an application potential for SLBs as very precise, nanoscale hydration sensors.

Antibodies to myelin oligodendrocyte glycoprotein (MOG-Abs) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (P < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab')2 rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Förster resonance energy transfer signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-Ab associated disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with antibodies to aquaporin-4 ., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

One of the great challenges of bottom-up synthetic biology is to recreate the cellular geometry and surface functionality required for biological reactions. Of particular interest are lipid membrane interfaces where many protein functions take place. However, cellular 3D geometries are often complex, and custom-shaping stable lipid membranes on relevant spatial scales in the micrometer range has been hard to accomplish reproducibly. Here, we use two-photon direct laser writing to 3D print microenvironments with length scales relevant to cellular processes and reactions. We formed lipid bilayers on the surfaces of these printed structures, and we evaluated multiple combinatorial scenarios, where physiologically relevant membrane compositions were generated on several different polymer surfaces. Functional dynamic protein systems were reconstituted in vitro and their self-organization was observed in response to the 3D geometry. This method proves very useful to template biological membranes with an additional spatial dimension, and thus allows a better understanding of protein function in relation to the complex morphology of cells and organelles.

The MinDE proteins from E. coli have received great attention as a paradigmatic biological pattern-forming system. Recently, it has surfaced that these proteins do not only generate oscillating concentration gradients driven by ATP hydrolysis, but that they can reversibly deform giant vesicles. In order to explore the potential of Min proteins to actually perform mechanical work, we introduce a new model membrane system, flat vesicle stacks on top of a supported lipid bilayer. MinDE oscillations can repeatedly deform these flat vesicles into tubules and promote progressive membrane spreading through membrane adhesion. Dependent on membrane and buffer compositions, Min oscillations further induce robust bud formation. Altogether, we demonstrate that under specific conditions, MinDE self-organization can result in work performed on biomimetic systems and achieve a straightforward mechanochemical coupling between the MinDE biochemical reaction cycle and membrane transformation., (© 2020 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Membrane-active cytoskeletal elements, such as FtsZ, septin or actin, form filamentous polymers able to induce and stabilize curvature on cellular membranes. In order to emulate the characteristic dynamic self-assembly properties of cytoskeletal subunits in vitro, biomimetic synthetic scaffolds were here developed using DNA origami. In contrast to our earlier work with pre-curved scaffolds, we specifically assessed the potential of origami mimicking straight filaments, such as actin and microtubules, by origami presenting cholesteryl anchors for membrane binding and additional blunt end stacking interactions for controllable polymerization into linear filaments. By assessing the interaction of our DNA nanostructures with model membranes using fluorescence microscopy, we show that filaments can be formed, upon increasing MgCl2 in solution, for structures displaying blunt ends; and can subsequently depolymerize, by decreasing the concentration of MgCl2. Distinctive spike-like membrane protrusions were generated on giant unilamellar vesicles at high membrane-bound filament densities, and the presence of such deformations was reversible and shown to correlate with the MgCl2-triggered polymerization of DNA origami subunits into filamentous aggregates. In the end, our approach reveals the formation of membrane-bound filaments as a minimal requirement for membrane shaping by straight cytoskeletal-like objects.

As a key mechanism underpinning many biological processes, protein self-organization has been extensively studied. However, the potential to apply the distinctive, nonlinear biochemical properties of such self-organizing systems to biotechnological problems such as the facile detection and characterization of biomolecular interactions has not yet been explored. Here, we describe an in vitro assay in a 96-well plate format that harnesses the emergent behavior of the Escherichia coli Min system to provide a readout of biomolecular interactions. Crucial for the development of our approach is a minimal MinE-derived peptide that stimulates MinD ATPase activity only when dimerized. We found that this behavior could be induced via any pair of foreign, mutually binding molecular entities fused to the minimal MinE peptide. The resulting MinD ATPase activity and the spatiotemporal nature of the produced protein patterns quantitatively correlate with the affinity of the fused binding partners, thereby enabling a highly sensitive assay for biomolecular interactions. Our assay thus provides a unique means of quantitatively visualizing biomolecular interactions and may prove useful for the assessment of domain interactions within protein libraries and for the facile investigation of potential inhibitors of protein-protein interactions.

Giant unilamellar phospholipid vesicles are attractive starting points for constructing minimal living cells from the bottom-up. Their membranes are compatible with many physiologically functional modules and act as selective barriers, while retaining a high morphological flexibility. However, their spherical shape renders them rather inappropriate to study phenomena that are based on distinct cell shape and polarity, such as cell division. Here, a microscale device based on 3D printed protein hydrogel is introduced to induce pH-stimulated reversible shape changes in trapped vesicles without compromising their free-standing membranes. Deformations of spheres to at least twice their aspect ratio, but also toward unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations, from the recurrent formation of dumbbells with curved necks between domains to full budding of membrane domains as separate vesicles. Moreover, shape-tunable vesicles are particularly desirable when reconstituting geometry-sensitive protein networks, such as reaction-diffusion systems. In particular, vesicle shape changes allow to switch between different modes of self-organized protein oscillations within, and thus, to influence reaction networks directly by external mechanical cues., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In bottom-up synthetic biology, one of the major methodological challenges is to provide reaction spaces that mimic biological systems with regard to topology and surface functionality. Of particular interest are cell- or organelle-shaped membrane compartments, as many protein functions unfold at lipid interfaces. However, shaping artificial cell systems using materials with non-intrusive physicochemical properties, while maintaining flexible lipid interfaces relevant to the reconstituted protein systems, is not straightforward. Herein, we develop micropatterned chambers from CYTOP, a less commonly used polymer with good chemical resistance and a refractive index matching that of water. By forming a self-assembled lipid monolayer on the polymer surface, we dramatically increased the biocompatibility of CYTOP-fabricated systems. The phospholipid interface provides an excellent passivation layer to prevent protein adhesion to the hydrophobic surface, and we succeeded in cell-free protein synthesis inside the chambers. Importantly, the chambers could be sealed after loading by a lipid monolayer, providing a novel platform to study encapsulated systems. We successfully reconstituted pole-to-pole oscillations of the Escherichia coli MinDE system, which responds dramatically to compartment geometry. Furthermore, we present a simplified fabrication of our artificial cell compartments via replica molding, making it a readily accessible technique for standard cleanroom facilities.

Reproduction, i.e. the ability to produce new individuals from a parent organism, is a hallmark of living matter. Even the simplest forms of reproduction require cell division: attempts to create a designer cell therefore should include a synthetic cell division machinery. In this review, we will illustrate how nature solves this task, describing membrane remodelling processes in general and focusing on bacterial cell division in particular. We discuss recent progress made in their in vitro reconstitution, identify open challenges, and suggest how purely synthetic building blocks could provide an additional and attractive route to creating artificial cell division machineries.

Ceramides are central intermediates of sphingolipid metabolism that also function as potent messengers in stress signaling and apoptosis. Progress in understanding how ceramides execute their biological roles is hampered by a lack of methods to manipulate their cellular levels and metabolic fate with appropriate spatiotemporal precision. Here, we report on clickable, azobenzene-containing ceramides, caCers, as photoswitchable metabolic substrates to exert optical control over sphingolipid production in cells. Combining atomic force microscopy on model bilayers with metabolic tracing studies in cells, we demonstrate that light-induced alterations in the lateral packing of caCers lead to marked differences in their metabolic conversion by sphingomyelin synthase and glucosylceramide synthase. These changes in metabolic rates are instant and reversible over several cycles of photoswitching. Our findings disclose new opportunities to probe the causal roles of ceramides and their metabolic derivatives in a wide array of sphingolipid-dependent cellular processes with the spatiotemporal precision of light., Competing Interests: MK, BW, HT, HF, SK, CS, PS, DT, JH, JF No competing interests declared, (© 2019, Kol et al.)

DNA origami nanotechnology is being increasingly used to mimic membrane-associated biophysical phenomena. Although a variety of DNA origami nanostructures has already been produced to target lipid membranes, the requirements for membrane binding have so far not been systematically assessed. Here, we used a set of elongated DNA origami structures with varying placement and number of cholesteryl-based membrane anchors to compare different strategies for their incorporation. Single and multiple cholesteryl anchors were attached to DNA nanostructures using single- and double-stranded DNA spacers of varying length. The produced DNA nanostructures were studied in terms of their membrane binding and diffusion. Our results show that the location and number of anchoring moieties play a crucial role for membrane binding of DNA nanostructures mainly if the cholesteryl anchors are in close proximity to the bulky DNA nanostructures. Moreover, the use of DNA spacers largely overcomes local steric hindrances and thus enhances membrane binding. Fluorescence correlation spectroscopy measurements demonstrate that the distinct physical properties of single- and double-stranded DNA spacers control the interaction of the amphipathic DNA nanostructures with lipid membranes. Thus, we provide a rational basis for the design of amphipathic DNA origami nanostructures to efficiently bind lipid membranes in various environments.

Membrane sculpting and transformation is essential for many cellular functions, thus being largely regulated by self-assembling and self-organizing protein coats. Their functionality is often encoded by particular spatial structures. Prominent examples are BAR domain proteins, the 'banana-like' shapes of which are thought to aid scaffolding and membrane tubulation. To elucidate whether 3D structure can be uncoupled from other functional features of complex scaffolding proteins, we hereby develop curved DNA origami in various shapes and stacking features, following the presumable design features of BAR proteins, and characterize their ability for membrane binding and transformation. We show that dependent on curvature, membrane affinity and surface density, DNA origami coats can indeed reproduce the activity of membrane-sculpting proteins such as BAR, suggesting exciting perspectives for using them in bottom-up approaches towards minimal biomimetic cellular machineries.

The endosomal sorting complex required for transport (ESCRT)-III is critical for membrane abscission; however, the mechanism underlying ESCRT-III-mediated membrane constriction remains elusive. A study of the dynamic assembly and disassembly of the ESCRT-III machinery in vitro and in vivo now suggests that the turnover of the observed spiralling filaments is critical for membrane abscission during cytokinesis.

Ceramide is a pro-apoptotic sphingolipid with unique physical characteristics. Often viewed as a second messenger, its generation can modulate the structure of lipid rafts. We prepared three photoswitchable ceramides, ACes, which contain an azobenzene photoswitch allowing for optical control over the N-acyl chain. Using combined atomic force and confocal fluorescence microscopy, we demonstrate that the ACes enable reversible switching of lipid domains in raft-mimicking supported lipid bilayers (SLBs). In the trans-configuration, the ACes localize into the liquid-ordered (L o ) phase. Photoisomerization to the cis-form triggers a fluidification of the L o domains, as liquid-disordered (L d ) "lakes" are formed within the rafts. Photoisomerization back to the trans-state with blue light stimulates a rigidification inside the L d phase, as the formation of small L o domains. These changes can be repeated over multiple cycles, enabling a dynamic spatiotemporal control of the lipid raft structure with light.

Over the last decade, functionally designed DNA nanostructures applied to lipid membranes prompted important achievements in the fields of biophysics and synthetic biology. Taking advantage of the universal rules for self-assembly of complementary oligonucleotides, DNA has proven to be an extremely versatile biocompatible building material on the nanoscale. The possibility to chemically integrate functional groups into oligonucleotides, most notably with lipophilic anchors, enabled a widespread usage of DNA as a viable alternative to proteins with respect to functional activity on membranes. As described throughout this review, hybrid DNA-lipid nanostructures can mediate events such as vesicle docking and fusion, or selective partitioning of molecules into phase-separated membranes. Moreover, the major benefit of DNA structural constructs, such as DNA tiles and DNA origami, is the reproducibility and simplicity of their design. DNA nanotechnology can produce functional structures with subnanometer precision and allow for a tight control over their biochemical functionality, e.g., interaction partners. DNA-based membrane nanopores and origami structures able to assemble into two-dimensional networks on top of lipid bilayers are recent examples of the manifold of complex devices that can be achieved. In this review, we will shortly present some of the potentially most relevant avenues and accomplishments of membrane-anchored DNA nanostructures for investigating, engineering, and mimicking lipid membrane-related biophysical processes., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

The DNA binding proteins from starved cells from Deinococcus radiodurans, Dps1-DR2263 and Dps2-DRB0092, have a common overall structure of hollow spherical dodecamers. Their involvement in the homeostasis of intracellular metal and DNA protection was addressed. Our results show that DrDps proteins are able to oxidize ferrous to ferric iron by oxygen or hydrogen peroxide. The iron stored inside the hollow sphere cavity is fully released. Furthermore, these proteins are able to store and release manganese, suggesting they can play a role in manganese homeostasis as well. The interaction of DrDps with DNA was also addressed. Even though DrDps1 binds both linear and coiled DNA, DrDps2 preferentially binds to coiled DNA, forming different protein-DNA complexes, as clearly shown by atomic force microscopy. DrDps1 (dimer and dodecamer) and DrDps2 can protect DNA against reactive oxygen species, although the protection occurs at different Fe to protein ratios. The difference between DrDps could be the result of the DrDps1 higher iron oxidation rate in the presence of hydrogen peroxide and its higher affinity to bind DNA than in DrDps2. Using cellular extracts obtained from D. radiodurans cultures, we showed that DrDps1 oligomers observed in in vitro conditions are also present in vivo. This indicates that DrDps1 has a structural dynamic plasticity that allows its oligomeric state to change between dimer, trimer and dodecamer. This in turn suggests the existence of a regulation mechanism that modulates the oligomer equilibrium and is dependent on growth stages and environmental conditions., (© 2015 FEBS.)

Amyloid fibers, implicated in a wide range of diseases, are formed when proteins misfold and stick together in long rope-like structures. As a natural mechanism, osmolytes can be used to modulate protein aggregation pathways with no interference with other cellular functions. The osmolyte sucrose delays fibrillation of the ribosomal protein S6 leading to softer and less shaped-defined fibrils. The molecular mechanism used by sucrose to delay S6 fibrillation was studied based on the two-state unfolding kinetics of the secondary and tertiary structures. It was concluded that the delay in S6 fibrillation results from stabilization and compaction of the slightly expanded tertiary native structure formed under fibrillation conditions. Interestingly, this compaction extends to almost all S6 tertiary structure but hardly affects its secondary structure. The part of the S6 tertiary structure that suffered more compaction by sucrose is known to be the first part to unfold, indicating that the native S6 has entered the unfolding pathway under fibrillation conditions., (© 2015 Wiley Periodicals, Inc.)

We report a synthetic biology-inspired approach for the engineering of amphipathic DNA origami structures as membrane-scaffolding tools. The structures have a flat membrane-binding interface decorated with cholesterol-derived anchors. Sticky oligonucleotide overhangs on their side facets enable lateral interactions leading to the formation of ordered arrays on the membrane. Such a tight and regular arrangement makes our DNA origami capable of deforming free-standing lipid membranes, mimicking the biological activity of coat-forming proteins, for example, from the I-/F-BAR family., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

New classes of antibiotics, such as antimicrobial peptides or proteins (AMPs), are crucial to deal with threatening bacterial diseases. rBPI21 is an AMP based on the human neutrophil BPI protein, with potential clinical use against meningitis. We studied the membrane perturbations promoted by rBPI21 on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Its interaction with bacteria was also studied in the presence of lipopolysaccharide (LPS), rBPI21 major ligand. Flow cytometry analysis of both bacteria shows that rBPI21 induces membrane depolarization. rBPI21 increases the negative surface charge of both bacteria toward positive values, as shown by zeta-potential measurements. This is followed by surface perturbations, culminating in cell lysis, as visualized by atomic force microscopy (AFM). Force spectroscopy measurements show that soluble LPS decreases the interaction of rBPI21 with bacteria, especially with S. aureus. This suggests that the rBPI21 LPS-binding pocket may also participate on the binding to Gram-positive bacteria., From the Clinical Editor: In this study, rBPI21, an antimicrobial protein based on the human neutrophil BPI protein, with potential clinical use against meningitis, is analyzed with multiple tools including zeta-potential measurements, clarifying its actions on E. coli and S. aureus. Since antimicrobial peptides are potentially important new additions to antibiotic regimens, studies like this represent important cornerstones in efficiency and mechanism of action testing of these new approaches., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Autophagy is a catabolic pathway that sequesters undesired cellular material into autophagosomes for delivery to lysosomes for degradation. A key step in the pathway is the covalent conjugation of the ubiquitin-related protein Atg8 to phosphatidylethanolamine (Atg8-PE) in autophagic membranes by a complex consisting of Atg16 and the Atg12-Atg5 conjugate. Atg8 controls the expansion of autophagic precursor membranes, but the underlying mechanism remains unclear. Here, we reconstitute Atg8 conjugation on giant unilamellar vesicles and supported lipid bilayers. We found that Atg8-PE associates with Atg12-Atg5-Atg16 into a membrane scaffold. By contrast, scaffold formation is counteracted by the mitochondrial cargo adaptor Atg32 through competition with Atg12-Atg5 for Atg8 binding. Atg4, previously known to recycle Atg8 from membranes, disassembles the scaffold. Importantly, mutants of Atg12 and Atg16 deficient in scaffold formation in vitro impair autophagy in vivo. This suggests that autophagic scaffolds are critical for phagophore biogenesis and thus autophagy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ζ potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

HCV NS4B, a highly hydrophobic protein involved in the alteration of the intracellular host membranes forming the replication complex, plays a critical role in the HCV life cycle. NS4B is a multifunctional membrane protein that possesses different regions where diverse and significant functions are located. One of these important regions is the AH2 segment, which besides being highly conserved has been shown to play a significant role in NS4B functioning. We have carried out an in-depth biophysical study aimed at the elucidation of the capacity of this region to interact, modulate and disrupt membranes, as well as to study the structural and dynamic features relevant for that disruption. We show that a peptide derived from this region, NS4BAH2, is capable of specifically binding phosphatidyl inositol phosphates with high affinity, and its interfacial properties suggest that this segment could behave similarly to a pre-transmembrane domain partitioning into and interacting with the membrane depending on the membrane composition and/or other proteins. Moreover, NS4BAH2 is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. NS4BAH2 is located in a shallow position in the membrane but it is able to affect the lipid environment from the membrane surface down to the hydrophobic core. The NS4B region where peptide NS4BAH2 resides might have an essential role in the membrane replication and/or assembly of the viral particle through the modulation of the membrane structure and hence the replication complex., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15μM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides., (Copyright © 2013 Elsevier B.V. All rights reserved.)

BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. Hydrogels composed of self-assembling β-hairpin peptides, having a high content of arginine, were found to be extremely effective at killing both gram-positive and gram-negative bacteria, including multi-drug resistant Pseudomonas aeruginosa. No added antibacterial agents are necessary to realize activity. Using self-assembling peptides for material construction allows facile structure-activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material's antibacterial properties. SAR studies show that arginine content largely influences the hydrogel's antibacterial activity, and influences their bulk rheological properties. These studies culminated in an optimized gel, composed of the peptide PEP6R (VKVRVRVRV(D)PPTRVRVRVKV). PEP6R gels prepared at 1.5 wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible toward human erythrocytes as well as mammalian mesenchymal stem cells. Rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe., (Published by Elsevier Ltd.)

Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH(2), IbKTP, IbKTP-NH(2) - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cyclotides, a large family of cyclic peptides from plants, have a broad range of biological activities, including insecticidal, cytotoxic, and anti-HIV activities. In all of these activities, cell membranes seem likely to be the primary target for cyclotides. However, the mechanistic role of lipid membranes in the activity of cyclotides remains unclear. To determine the role of lipid organization in the activity of the prototypic cyclotide, kalata B1 (kB1), and synthetic analogs, their bioactivities and affinities for model membranes were evaluated. We found that the bioactivity of kB1 is dependent on the lipid composition of target cell membranes. In particular, the activity of kB1 requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups but is further modulated by nonspecific peptide-lipid hydrophobic interactions, which are favored in raft-like membranes. Negatively charged phospholipids do not favor high kB1 affinity. This lipid selectivity explains trends in antimicrobial and hemolytic activities of kB1; it does not target bacterial cell walls, which are negatively charged and lacking PE-phospholipids but can insert in the membranes of red blood cells, which have a low PE content and raft domains in their outer layer. We further show that the anti-HIV activity of kB1 is the result of its ability to target and disrupt the membranes of HIV particles, which are raft-like membranes very rich in PE-phospholipids.

Objectives: 2F5 and 4E10 are two broadly neutralizing monoclonal antibodies (mAbs) targeting the membrane proximal external region (MPER) of HIV-1 gp41 envelope protein. This region, which contacts the viral membrane, is highly conserved and has been regarded as a promising target for vaccine development. We aimed to clarify the basis of 2F5 and 4E10 molecular interactions with epitope cores in MPER and lipid bilayers., Design: Microscopy-based approaches were used to infer and quantify the effects of both mAbs on membranes, in the presence and absence of the epitope cores. Supported lipid bilayers (SLBs), with and without phase separation, were used as membrane models. Fluorescent-labeled and nonlabeled MPER-derived peptides containing both 2F5 and 4E10 epitopes were used., Methods: mAbs 2F5 and 4E10 membrane interactions, in the presence or absence of MPER-derived peptides, were evaluated by combined atomic force and confocal microscopies., Results: Both mAbs form lipid-segregated aggregates on SLBs and do not induce other significant membrane perturbations. Furthermore, the affinity of MPER toward membranes is differently affected by both mAbs and correlates with the mAbs-epitope core lipid interactions. 2F5 is able to dock the MPER peptide on the membrane, whereas 4E10 extracts the MPER from the lipid bilayer., Conclusion: The results reveal the molecular details underneath 2F5/4E10 membrane-epitope binding and a model is proposed to explain the differential mAbs neutralization efficacies, which relates to the exposure of the epitopes in the lipid bilayers and the role of the lipids in mAb-epitope binding.

Peptide-membrane interactions have been gaining increased relevance, mainly in biomedical investigation, as the potential of the natural, nature-based and synthetic peptides as new drugs or drug candidates also expands. These peptides must face the cell membrane when they interfere with or participate in intracellular processes. Additionally, several peptide drugs and drug leads actions occur at the membrane level (e.g., antimicrobial peptides, cell-penetrating peptides and enveloped viruses membrane fusion inhibitors). Here we explore fluorescence spectroscopy methods that can be used to monitor such interactions. Two main approaches are considered, centered either on the peptide or on the membrane. On the first, we consider mainly the methodologies based on the intrinsic fluorescence of the aminoacid residues tryptophan and tyrosine. Regarding membrane-centric approaches, we review methods based on lipophilic probes sensitive to membrane potentials. The use of fluorescence constitutes a simple and sensitive method to measure these events. Unraveling the molecular mechanisms that govern these interactions can unlock the key to understand specific biological processes involving natural peptides or to optimize the action of a peptide drug., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The potential of antimicrobial peptides (AMPs) as an alternative to conventional therapies is well recognized. Insights into the biological and biophysical properties of AMPs are thus key to understanding their mode of action. In this study, the mechanisms adopted by two AMPs in disrupting the gram-negative Escherichia coli bacterial envelope were explored. BP100 is a short cecropin A-melittin hybrid peptide known to inhibit the growth of phytopathogenic gram-negative bacteria. pepR, on the other hand, is a novel AMP derived from the dengue virus capsid protein. Both BP100 and pepR were found to inhibit the growth of E. coli at micromolar concentrations. Zeta potential measurements of E. coli incubated with increasing peptide concentrations allowed for the establishment of a correlation between the minimal inhibitory concentration (MIC) of each AMP and membrane surface charge neutralization. While a neutralization-mediated killing mechanism adopted by either AMP is not necessarily implied, the hypothesis that surface neutralization occurs close to MIC values was confirmed. Atomic force microscopy (AFM) was then employed to visualize the structural effect of the interaction of each AMP with the E. coli cell envelope. At their MICs, BP100 and pepR progressively destroyed the bacterial envelope, with extensive damage already occurring 2 h after peptide addition to the bacteria. A similar effect was observed for each AMP in the concentration-dependent studies. At peptide concentrations below MIC values, only minor disruptions of the bacterial surface occurred.

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel HIV-1 fusion inhibitor with improved antiretroviral activity. In this work we evaluated the physical chemistry foundation of the interaction of sifuvirtide with biomembrane model systems. Since this peptide has aromatic residues, fluorescence spectroscopy techniques were mostly used. The interaction was assessed by partition and quenching experiments. Results showed no significant interaction with large unilamellar vesicles composed by sphingomyelin and ceramide. In contrast, sifuvirtide presented selectivity towards vesicles composed by phosphatidylcholines (PC) in the gel phase, in opposition to fluid phase PC vesicles. The interaction of this peptide with gel phase PC membranes (K(p)=1.2x10(2)) is dependent on the ionic strength, which indicates the mediation of electrostatic interactions at an interfacial level. The effects of sifuvirtide on the lipid membranes' structural properties were further evaluated using dipole-potential membrane probes, zeta-potential, dynamic light scattering and atomic force microscopy measurements. The results show that sifuvirtide does not cause a noticeable effect on lipid bilayer structure, except for membranes composed by cationic phospholipids. Altogether, we can conclude that sifuvirtide presents a specific affinity towards rigid PC membranes, and the interaction is mediated by electrostatic factors, not affecting the membrane architecture.

Sifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion inhibitor, presently in clinical trials. Because of the aromatic amino acid residues of the peptide, its behavior in aqueous solution and the interaction with lipid-membrane model systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy techniques (both steady-state and time-resolved). No significant aggregation of the peptide was observed with aqueous solution. Various biological and nonbiological lipid-membrane compositions were analyzed, and atomic force microscopy was used to visualize phase separation in several of those mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquid-ordered phase). However, significant partitioning was observed with the positively charged lipid models (K(p) = (2.2 +/- 0.3) x 10(3)), serving as a positive control. Fluorescence quenching using Förster resonance acrylamide and lipophilic probes was carried out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at the surface of these membranes was observed and confirmed by using Förster resonance energy-transfer experiments. These results indicate a targeting of the peptide to gel-phase domains relatively to liquid-disordered or liquid-ordered phase domains. This larger affinity and selectivity toward the more rigid areas of the membranes, where most of the receptors are found, or to viral membrane, may help explain the improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the peptide at the fusion site.

Protein-protein interactions, as well as peptide-peptide and peptide-protein interactions are fields of study of growing importance as molecular-level detail is avidly pursued in drug design, metabolic regulation and molecular dynamics, among other classes of studies. In membranes, this issue is particularly relevant because lipid bilayers potentiate molecular interactions due to the high local concentration of peptides and other solutes.However, experimental techniques and methodologies to detect and quantify such interactions are not abundant. A reliable, fast and inexpensive alternative methodology is revisited in this work. Considering the interaction of two molecules, at least one of them being fluorescent, either intrinsically (e.g. Trp residues) or by grafting a specific probe, changes in their aggregation state may be reported, as long as the fluorophore is sensitive to local changes in polarity, conformation and/or exposure to the solvent. The interaction will probably lead to modifications in fluorescence intensity resulting in a decrease ('quenching') or enhancement ('dequenching'). Although the presented methodology is based on static quenching methodologies, the concept is extended from quenching to any kind of interference with the fluorophore. Equations for data analysis are shown and their applications are illustrated by calculating the binding constant for several data-sets.