Parkinson's disease-associated mutations in α-synuclein alters its lipid-bound state.

Lipid-binding properties of α-synuclein play a central role in protein aggregation and progression of Parkinson's disease (PD). α-Synuclein, an intrinsically disordered protein, binds to lipid membranes through the formation of two amphipathic helices that insert into the lipid bilayer. All disease-associated single point mutations have been identified to be within these helical regions of α-synuclein: V15A, A30P, E46K, H50Q, G51D, A53T, and A53V. However, the effects of these mutations on the bound states of the two α helices of the protein have yet to be fully characterized. In this report, we use a tryptophan fluorescence assay to measure the binding of the α helices of these PD-associated mutants to lipid membranes within the lipid-depletion regime. We characterize the binding behavior of each helix, revealing that, generally, the PD-associated mutants shift the equilibrium bound state away from the N-terminal helix of the protein toward helix 2 at lower lipid concentrations. Altogether, our results indicate that disruption to the equilibrium binding of the two α helices of α-synuclein could play a role in PD progression.

Binding equations for the lipid composition dependence of peripheral membrane-binding proteins.

The specific recognition of peripheral membrane-binding proteins for their target membranes is mediated by a complex constellation of various lipid contacts. Despite the inherent complexities of the heterogeneous protein-membrane interface, the binding dependence of such proteins is, surprisingly, often reliably described by simple models such as the Langmuir Adsorption Isotherm or the Hill equation. However, these models were not developed to describe associations with two-dimensional, highly concentrated heterogeneous ligands such as lipid membranes. In particular, these models fail to capture the dependence on the lipid composition, a significant determinant of binding that distinguishes target from non-target membranes. In this work, we present a model that describes the dependence of peripheral proteins on lipid composition through an analytic expression for their association. The resulting membrane-binding equation retains the features of these simple models but completely describes the binding dependence on multiple relevant variables in addition to the lipid composition, such as protein and vesicle concentration. Implicit in this lipid composition dependence is a new form of membrane-based cooperativity that significantly differs from traditional solution-based cooperativity. We introduce the Membrane-Hill number as a measure of this cooperativity and describe its unique properties. We illustrate the utility and interpretational power of our model by analyzing previously published data on two peripheral proteins that associate with phosphatidylserine-containing membranes: The transmembrane immunoglobulin and mucin domain-containing protein 3 (TIM3) that employs calcium in its association, and milk fat globulin epidermal growth factor VIII (MFG-E8) which is completely insensitive to calcium. We also provide binding equations for systems that exhibit more complexity in their membrane-binding.

MFG-E8: a model of multiple binding modes associated with ps-binding proteins.

Membrane-binding proteins often associate with lipid membranes through a singular binding interface which is generally modeled as a two-state system: bound or unbound. However, even a single interface can engage with more than one mode of binding since a variety of interactions can contribute to the binding event. Unfortunately, the ability to clearly delineate the different binding modes of a singular binding interface has been elusive with existing models. Here, we present a study on milk fat globule EGF factor 8 (MFG-E8), which belongs to a class of proteins that identifies and binds phosphatidylserine (PS). These proteins detect membrane dysregulation implicated in exposed PS in apoptosis and malignant cells. In order to elucidate the factors affecting the binding of MFG-E8, we used a model system consisting of a series of lipid vesicles with varying PS mole fraction to identify the sensitivity of MFG-E8's binding affinity to changes in electrostatics using a tryptophan fluorescence spectral shift assay. Using a newly developed model, we experimentally identified three binding modes, each associated with a different number of PS lipids, with its cooperativity for binding being enhanced by the availability of negatively charged lipids. X-ray reflectivity experiments additionally suggest that MFG-E8's binding modes are influenced by membrane packing. The protocols established for elucidating MFG-E8's interaction with lipid membranes under different membrane conditions can be applied to the study of other membrane-binding proteins that target specific membrane attributes, such as fluidity and electrostatics, and help elucidate these membrane targeting mechanisms and their subsequent binding events.

How Tim proteins differentially exploit membrane features to attain robust target sensitivity.

Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.

Tailoring Biomimetic Phosphorylcholine-Containing Block Copolymers as Membrane-Targeting Cellular Rescue Agents.

Some synthetic polymers can block cell death when applied following an injury that would otherwise kill the cell. This cellular rescue occurs through interactions of the polymers with cell membranes. However, general principles for designing synthetic polymers to ensure strong, but nondisruptive, cell membrane targeting are not fully elucidated. Here, we tailored biomimetic phosphorylcholine-containing block copolymers to interact with cell membranes and determined their efficacy in blocking neuronal death following oxygen-glucose deprivation. By adjusting the hydrophilicity and membrane affinity of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC)-based triblock copolymers, the surface active regime in which the copolymers function effectively as membrane-targeting cellular rescue agents was determined. We identified nonintrusive interactions between the polymer and the cell membrane that alter the collective dynamics of the membrane by inducing rigidification without disrupting lipid packing or membrane thickness. In general, our results open new avenues for biological applications of polyMPC-based polymers and provide an approach to designing membrane-targeting agents to block cell death after injury.

Impact of Surface Amphiphilicity on the Interfacial Behavior of Janus Particle Layers under Compression.

Langmuir monolayers of silica/gold Janus particles with two different degrees of amphiphilicity have been examined to study the significance of particle surface amphiphilicity on the structure and mechanical properties of the interfacial layers. The response of the layers to the applied compression provides insight into the nature and strength of the interparticle interactions. Different collapse modes observed for the interfacial layers are linked to the amphiphilicity of Janus particles and their configuration at the interface. Molecular dynamics simulations on nanoparticles with similar contact angles provide insight on the arrangement of particles at the interface and support our conclusion that the interfacial configuration and collapse of anisotropic particles at the air/water interface are controlled by particle amphiphilicity.

Beyond electrostatics: Antimicrobial peptide selectivity and the influence of cholesterol-mediated fluidity and lipid chain length on protegrin-1 activity.

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1's incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.

α-Synuclein Sterically Stabilizes Spherical Nanoparticle-Supported Lipid Bilayers.

While it is generally accepted that neuronal protein α-synuclein binds to highly curved and highly charged lipid membranes, its biological function beyond binding remains unknown despite its fundamental link to Parkinson's disease. Herein, we utilize spherical nanoparticle lipid bilayers (SSLBs) to recapitulate the charge and curvature limit of membrane organelles with which α-synuclein associates and probe how α-synuclein affects SSLB structure and dynamics as a proxy for interorganelle interactions. Small-angle X-ray scattering and X-ray photon correlation spectroscopy reveal our SSLBs form aggregates that are clearly broken up by the addition of α-synuclein, a clear indication that α-synuclein confers steric stabilization to membrane surfaces.

Osmotic Shock-Triggered Assembly of Highly Charged, Nanoparticle-Supported Membranes.

Spherical nanoparticle-supported lipid bilayers (SSLBs) combine precision nanoparticle engineering with biocompatible interfaces for various applications, ranging from drug delivery platforms to structural probes for membrane proteins. Although the bulk, spontaneous assembly of vesicles and larger silica nanoparticles (>100 nm) robustly yields SSLBs, it will only occur with low charge density vesicles for smaller nanoparticles (<100 nm), a fundamental barrier in increasing SSLB utility and efficacy. Here, through whole mount and cryogenic transmission electron microscopy, we demonstrate that mixing osmotically loaded vesicles with smaller nanoparticles robustly drives the formation of SSLBs with high membrane charge density (up to 60% anionic lipid or 50% cationic lipid). We show that the osmolyte load necessary for SSLB formation is primarily a function of absolute membrane charge density and is not lipid headgroup-dependent, providing a generalizable, tunable approach toward bulk production of highly curved and charged SSLBs with various membrane compositions.

Sensitivity of peripheral membrane proteins to the membrane context: A case study of phosphatidylserine and the TIM proteins.

There is a diverse class of peripheral membrane-binding proteins that specifically bind phosphatidylserine (PS), a lipid that signals apoptosis or cell fusion depending on the membrane context of its presentation. PS-receptors are specialized for particular PS-presenting pathways, indicating that they might be sensitive to the membrane context. In this review, we describe a combination of thermodynamic, structural, and computational techniques that can be used to investigate the mechanisms underlying this sensitivity. As an example, we focus on three PS-receptors of the T-cell Immunoglobulin and Mucin containing (TIM) protein family, which we have previously shown to differ in their sensitivity to PS surface density.

Enhanced Ordering in Monolayers Containing Glycosphingolipids: Impact of Carbohydrate Structure.

The influence of carbohydrate structure on the ordering of glycosphingolipids (GSLs) and surrounding phospholipids was investigated in monolayers at the air-water interface. Binary mixtures composed of GSLs, chosen to span a range of carbohydrate complexity, and zwitterionic dipalmitoylphosphatidylcholine phospholipid, were studied. X-ray reflectivity was used to measure the out-of-plane structure of the monolayers and characterize the extension and conformation of the GSL carbohydrates. Using synchrotron grazing incidence x-ray diffraction, the in-plane packing of the lipid acyl chains and the area per molecule within ordered domains were characterized at different mole ratios of the two components. Our findings indicate that GSL-containing mixtures, regardless of the carbohydrate size, enhance the ordering of the surrounding lipids, resulting in a larger fraction of ordered phase of the monolayer and greater dimensions of the ordered domains. Reduction of the averaged area per molecule within the ordered domains was also observed but only in the cases where there was a size mismatch between the phospholipid headgroups and GSL components, suggesting that the condensation mechanism involves the relief of steric interactions between headgroups in mixtures.

Coupling X-Ray Reflectivity and In Silico Binding to Yield Dynamics of Membrane Recognition by Tim1.

The dynamic nature of lipid membranes presents significant challenges with respect to understanding the molecular basis of protein/membrane interactions. Consequently, there is relatively little known about the structural mechanisms by which membrane-binding proteins might distinguish subtle variations in lipid membrane composition and/or structure. We have previously developed a multidisciplinary approach that combines molecular dynamics simulation with interfacial x-ray scattering experiments to produce an atomistic model for phosphatidylserine recognition by the immune receptor Tim4. However, this approach requires a previously determined protein crystal structure in a membrane-bound conformation. Tim1, a Tim4 homolog with distinct differences in both immunological function and sensitivity to membrane composition, was crystalized in a closed-loop conformation that is unlikely to support membrane binding. Here we have used a previously described highly mobile membrane mimetic membrane in combination with a conventional lipid bilayer model to generate a membrane-bound configuration of Tim1 in silico. This refined structure provided a significantly improved fit of experimental x-ray reflectivity data. Moreover, the coupling of the x-ray reflectivity analysis with both highly mobile membrane mimetic membranes and conventional lipid bilayer molecular dynamics simulations yielded a dynamic model of phosphatidylserine membrane recognition by Tim1 with atomic-level detail. In addition to providing, to our knowledge, new insights into the molecular mechanisms that distinguish the various Tim receptors, these results demonstrate that in silico membrane-binding simulations can remove the requirement that the existing crystal structure be in the membrane-bound conformation for effective x-ray reflectivity analysis. Consequently, this refined methodology has the potential for much broader applicability with respect to defining the atomistic details of membrane-binding proteins.

Quantitative analysis of total reflection X-ray fluorescence from finely layered structures using XeRay.

Total reflection x-ray fluorescence (TXRF) is a widely applicable experimental technique for studying chemical element distributions across finely layered structures at extremely high sensitivity. To promote and facilitate scientific discovery using TXRF, we developed a MATLAB-based software package with a graphical user interface, named XeRay, for quick, accurate, and intuitive data analysis. XeRay lets the user model any layered system, each layer with its independent chemical composition and thickness, and enables fine-tuned data fitting. The accuracy of XeRay has been tested in the analysis of TXRF data from both air/liquid interface and liquid/liquid interfacial studies and has been compared to literature results. In an air/liquid interface study, Ca2+ sequestration was measured at a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (SOPA) on a buffer solution of 1 mM CaCl2 at pH 7.5. Data analysis with XeRay reveals that each 1 nm2 of interfacial area contains 2.38 ± 0.06 Ca2+ ions, which corresponds to a 1:1 ratio between SOPA headgroups and Ca2+ ions, consistent with several earlier reports. For the liquid/liquid interface study of Sr2+ enrichment at the dodecane/surfactant/water interface, analysis using XeRay gives a surface enrichment of Sr2+ at 68-5+6 Å2 per ion, consistent with the result published for the same dataset.

Robust Gold Nanoparticle Sheets by Ligand Cross-Linking at the Air-Water Interface.

We report the results of cross-linking of two-dimensional gold nanoparticle (Au-NP) assemblies at the air-water interface in situ. We introduce an aqueous soluble ruthenium benzylidene catalyst into the water subphase to generate a robust, elastic two-dimensional network of nanoparticles containing cyclic olefins in their ligand framework. The most striking feature of the cross-linked Au-NP assemblies is that the extended connectivity of the nanoparticles enables the film to preserve much of its integrity under compression and expansion, features that are absent in its non-cross-linked counterparts. The cross-linking process appears to "stitch" the nanoparticle crystalline domains together, allowing the cross-linked monolayers to behave like a piece of fabric under lateral compression.

Long-Range Organization of Membrane-Curving Proteins.

Biological membranes have a central role in mediating the organization of membrane-curving proteins, a dynamic process that has proven to be challenging to probe experimentally. Using atomic force microscopy, we capture the hierarchically organized assemblies of Bin/amphiphysin/Rvs (BAR) proteins on supported lipid membranes. Their structure reveals distinct long linear aggregates of proteins, regularly spaced by up to 300 nm. Employing accurate free-energy calculations from large-scale coarse-grained computer simulations, we found that the membrane mediates the interaction among protein filaments as a combination of short- and long-ranged interactions. The long-ranged component acts at strikingly long distances, giving rise to a variety of micron-sized ordered patterns. This mechanism may contribute to the long-ranged spatiotemporal control of membrane remodeling by proteins in the cell.

Antimicrobial Peptides Share a Common Interaction Driven by Membrane Line Tension Reduction.

Antimicrobial peptides (AMPs) are a class of host-defense molecules that neutralize a broad range of pathogens. Their membrane-permeabilizing behavior has been commonly attributed to the formation of pores; however, with the continuing discovery of AMPs, many are uncharacterized and their exact mechanism remains unknown. Using atomic force microscopy, we previously characterized the disruption of model membranes by protegrin-1 (PG-1), a cationic AMP from pig leukocytes. When incubated with zwitterionic membranes of dimyristoylphosphocholine, PG-1 first induced edge instability at low concentrations, then porous defects at intermediate concentrations, and finally worm-like micelle structures at high concentrations. These rich structural changes suggested that pore formation constitutes only an intermediate state along the route of PG-1's membrane disruption process. The formation of these structures could be best understood by using a mesophase framework of a binary mixture of lipids and peptides, where PG-1 acts as a line-active agent in lowering interfacial bilayer tensions. We have proposed that rather than being static pore formers, AMPs share a common ability to lower interfacial tensions that promote membrane transformations. In a study of 13 different AMPs, we found that peptide line-active behavior was not driven by the overall charge, and instead was correlated with their adoption of imperfect secondary structures. These peptide structures commonly positioned charged residues near the membrane interface to promote deformation favorable for their incorporation into the membrane. Uniquely, the data showed that barrel-stave-forming peptides such as alamethicin are not line-active, and that the seemingly disparate models of toroidal pores and carpet activity are actually related. We speculate that this interplay between peptide structure and the distribution of polar residues in relation to the membrane governs AMP line activity in general and represents a novel, to our knowledge, avenue for the rational design of new drugs.

Liquid Surface X-ray Studies of Gold Nanoparticle-Phospholipid Films at the Air/Water Interface.

Amphiphilic phospholipids and nanoparticles functionalized with hydrophobic capping ligands have been extensively investigated for their capacity to self-assemble into Langmuir monolayers at the air/water interface. However, understanding of composite films consisting of both nanoparticles and phospholipids, and by extension, the complex interactions arising between nanomaterials and biological membranes, remains limited. In this work, dodecanethiol-capped gold nanoparticles (Au-NPs) with an average core diameter of 6 nm were incorporated into 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers with surface densities ranging from 0.1 to 20% area coverage at a surface pressure of 30 mN/m. High resolution liquid surface X-ray scattering studies revealed a phase separation of the DPPC and Au-NP components of the composite film, as confirmed with atomic force microscopy after the film was transferred to a substrate. At low Au-NP content, the structural organization of the phase-separated film is best described as a DPPC film containing isolated islands of Au-NPs. However, increasing the Au-NP content beyond 5% area coverage transforms the structural organization of the composite film to a long-range interconnected network of Au-NP strands surrounding small seas of DPPC, where the density of the Au-NP network increases with increasing Au-NP content. The observed phase separation and structural organization of the phospholipid and nanoparticle components in these Langmuir monolayers are useful for understanding interactions of nanoparticles with biological membranes.

Mechanical Stability of Polystyrene and Janus Particle Monolayers at the Air/Water Interface.

The compressional instability of particle-laden air/water interfaces is investigated with plain and surface-anisotropic (Janus) particles. We hypothesize that the amphiphilic nature of Janus particles leads to both anisotropic particle-particle and particle-interface interactions that can yield particle films with unique collapse mechanisms. Analysis of Langmuir isotherms and microstructural characterization of the homogeneous polystyrene particle films during compression reveal an interfacial buckling instability followed by folding, which is in good agreement with predictions from classical elasticity theory. In contrast, Janus particle films exhibit a different behavior during compression, where the collapse mode occurs through the subduction of the Janus particle film. Our results suggest that particle-laden films comprised of surface-anisotropic particles can be engineered to evolve new material properties.

Synergistic Interactions of Sugars/Polyols and Monovalent Salts with Phospholipids Depend upon Sugar/Polyol Complexity and Anion Identity.

We found that interactions of dipalmitoylphosphatidylcholine (DPPC) lipid monolayers with sugars are influenced by addition of NaCl. This work is of general importance in understanding how sugar-lipid-salt interactions impact biological systems. Using Langmuir isothermal compressions, fluorescence microscopy, atomic force microscopy, and neutron reflectometry, we examined DPPC monolayers upon addition of sugars/polyols and/or monovalent salts. Sugar-lipid interactions in the presence of NaCl increased with increasing complexity of the sugar/polyol in the order glycerol ≪ glucose < trehalose. When the anion was altered in the series NaF, NaCl, and NaBr, only minor differences were observed. When comparing LiCl, NaCl, and KCl, sodium chloride had the greatest influence on glucose and trehalose interactions with DPPC. We propose that heterogeneity created by cation binding allows for sugars to bind the lipid headgroups. While cation binding increases in the order K(+) < Na(+) < Li(+), lithium ions may also compete with glucose for binding sites. Thus, both cooperative and competitive factors contribute to the overall influence of salts on sugar-lipid interactions.

Collapse of Particle-Laden Interfaces under Compression: Buckling vs Particle Expulsion.

Colloidal particles can bind to fluid interfaces with a capillary energy that is thousands of times the thermal energy. This phenomenon offers an effective route to emulsion and foam stabilization where the stability is influenced by the phase behavior of the particle-laden interface under deformation. Despite the vast interest in particle-laden interfaces, the key factors that determine the collapse of such an interface under compression have remained relatively unexplored. In this study, we illustrate the significance of the particle surface wettability and presence of electrolyte in the subphase on interparticle interactions at the interface and the resulting collapse mode. Various collapse mechanisms including buckling, particle expulsion, and multilayer formation are reported and interpreted in terms of particle-particle and particle-interface interactions.