Lipid-Chaperone Hypothesis: A Common Molecular Mechanism of Membrane Disruption by Intrinsically Disordered Proteins.

An increasing number of human diseases has been shown to be linked to aggregation and amyloid formation by intrinsically disordered proteins (IDPs). Amylin, amyloid-ß, and α-synuclein are, indeed, involved in type-II diabetes, Alzheimer's, and Parkinson's, respectively. Despite the correlation of the toxicity of these proteins at early aggregation stages with membrane damage, the molecular events underlying the process is quite complex to understand. In this study, we demonstrate the crucial role of free lipids in the formation of lipid-protein complex, which enables an easy membrane insertion for amylin, amyloid-ß, and α-synuclein. Experimental results from a variety of biophysical methods and molecular dynamics results reveal that this common molecular pathway in membrane poration is shared by amyloidogenic (amylin, amyloid-ß, and α-synuclein) and nonamyloidogenic (rat IAPP, ß-synuclein) proteins. Based on these results, we propose a "lipid-chaperone" hypothesis as a unifying framework for protein-membrane poration.

Symmetry-breaking transitions in the early steps of protein self-assembly.

Protein misfolding and subsequent self-association are complex, intertwined processes, resulting in development of a heterogeneous population of aggregates closely related to many chronic pathological conditions including Type 2 Diabetes Mellitus and Alzheimer's disease. To address this issue, here, we develop a theoretical model in the general framework of linear stability analysis. According to this model, self-assemblies of peptides with pronounced conformational flexibility may become, under particular conditions, unstable and spontaneously evolve toward an alternating array of partially ordered and disordered monomers. The predictions of the theory were verified by atomistic molecular dynamics (MD) simulations of islet amyloid polypeptide (IAPP) used as a paradigm of aggregation-prone polypeptides (proteins). Simulations of dimeric, tetrameric, and hexameric human-IAPP self-assemblies at physiological electrolyte concentration reveal an alternating distribution of the smallest domains (of the order of the peptide mean length) formed by partially ordered (mainly ß-strands) and disordered (turns and coil) arrays. Periodicity disappears upon weakening of the inter-peptide binding, a result in line with the predictions of the theory. To further probe the general validity of our hypothesis, we extended the simulations to other peptides, the Aß(1-40) amyloid peptide, and the ovine prion peptide as well as to other proteins (SOD1 dimer) that do not belong to the broad class of intrinsically disordered proteins. In all cases, the oligomeric aggregates show an alternate distribution of partially ordered and disordered monomers. We also carried out Surface Enhanced Raman Scattering (SERS) measurements of hIAPP as an experimental validation of both the theory and in silico simulations.

Protein Adsorption at the Air-Water Interface by a Charge Sensing Interferometric Technique.

Protein uptake at the interface of a millimeter-sized air bubble in water is investigated by a recently developed differential interferometric technique. The technique allows the study of capillary waves with amplitudes around 10-9 m, excited at the surface of the bubble by an electric field of intensity on the order of 10 V/cm. When one studies the resonant modes of the bubble (radial and shape modes), it is possible to assess variations of interfacial properties and, in particular, of the net surface charge as a function of bulk protein concentration. Sensing the interfacial charge, the technique enables us to follow the absorption process in conditions of low concentrations, not easily assessable by other methods. We focus on bovine serum albumin (BSA) and lysozyme as representatives of typical globular proteins. To provide comprehensive insight into the novelty of the technique, we also investigated the equilibrium adsorption of sodium dodecyl sulfate (SDS) ionic surfactant for bulk concentrations at hundreds of times lower than the Critical Micelle Concentration (CMC). Results unveil how the absorption of charged molecules affects the amplitudes of the bubble resonant modes even before affecting the frequencies in a transition-like fashion. Different adsorption models are proposed and developed. They are validated against the experimental findings by comparing frequency and amplitude data. By measuring the charging rate of the bubble interface, we have followed the absorption kinetics of BSA and lysozyme recognizing a slow, energy barrier limited phenomena with characteristic times in agreement with data in the literature. The evaluation of the surface excess concentration (Γ) of BSA and SDS at equilibrium is obtained by monitoring charge uptake. At the investigated low bulk concentrations, reliable comparisons with literature data from equilibrium surface tension isotherm models are reported.

Helical Inclusions in Phospholipid Membranes: Lipid Adaptation and Chiral Order.

The lipid bilayer is a flexible matrix that is able to adapt in response to the perturbation induced by inclusions, such as peptides and proteins. Here we use molecular dynamics simulations with a coarse-grained model to investigate the effect of a helical inclusion on a lipid bilayer in the liquid disordered phase. We show that the helical inclusion induces a collective tilt of acyl chains, with a small, yet unambiguous difference between a right- and a left-handed inclusion. This behavior is rationalized using the elastic continuum theory: The magnitude of the chiral (twist) deformation of the bilayer is determined by the interaction at the lipid/inclusion interface, and the decay length is controlled by the elastic properties of the bilayer. The lipid reorganization can thus be identified as a generic mechanism that, together with specific interactions, contributes to chiral recognition in phospholipid bilayers. An enhanced response is expected in highly ordered environments, such as rafts in biomembranes, with a potential impact on membrane-mediated interactions between inclusions.

Phospholipids Critical Micellar Concentrations Trigger Different Mechanisms of Intrinsically Disordered Proteins Interaction with Model Membranes.

Amyloidogenic proteins are involved in many diseases, including Alzheimer's, Parkinson's, and type II diabetes. These proteins are thought to be toxic for cells because of their abnormal interaction with the cell membrane. Simpler model membranes (LUVs) have been used to study the early steps of membrane-protein interactions and their subsequent evolution. Phospholipid LUVs formed in water solution establish a chemical equilibrium between self-assembled LUVs and a small amount of phospholipids in water solution (CMC). Here, using both experimental and molecular dynamics simulations approach we demonstrate that the insertion of IAPP, an amyloidogenic peptide involved in diabetes, in membranes is driven by free lipids in solution in dynamic equilibrium with the self-assembled lipids of the bilayer. It is suggested that this could be a general mechanism lying at the root of membrane insertion processes of self-assembling peptides.

Nanometric Surface Oscillation Spectroscopy of Water-Poor Microemulsions.

Selectively exchanging metal complexes between emulsified water-poor microemulsions and concentrated solutions of mixed electrolytes is the core technology for strategic metal recycling. Nanostructuration triggered by solutes present in the organic phase is understood, but little is known about fluctuations of the microemulsion-water interface. We use here a modified version of an optoelectric device initially designed for air bubbles, in order to evidence resonant electrically induced surface waves of an oily droplet suspended in an aqueous phase. Resonant waves of nanometer amplitude of a millimeter-sized microemulsion droplet containing a common ion-specific extractant diluted by dodecane and suspended in a solution of rare earth nitrate are evidenced for the first time with low excitation fields (5 V/cm). From variation of the surface wave spectrum with rare earth concentration, we evidence uptake of rare-earth ions at the interface and at higher concentration the formation of a thin "crust" of liquid crystal forming at unusually low concentration, indicative of a surface induced phase transition. The effect of the liquid crystal structure on the resonance spectrum is backed up by a model, which is used to estimate crust thickness.

Chirality Enhancement of Porphyrin Supramolecular Assembly Driven by a Template Preorganization Effect.

Cationic polylysine promotes, under neutral conditions, the spontaneous aggregation of opposite charged ZnTPPS in water. Spectroscopic investigations evidence a different preorganization of ZnTPPS onto the polypeptide matrix depending on the chain length. Spinodal decomposition theory in confined geometry is used to model this mechanism by considering the time evolution of a homogeneous distribution of randomly adsorbed particles (porphyrins) onto a rodlike polyelectrolyte (polymer) of variable length L.

An interferometric technique to study capillary waves.

We describe a new interferometric technique to study gas-liquid and liquid-liquid interfaces. Bubbles and drops are subjected to an alternating electric field which excites capillary oscillations at the interface, if charged. Bubble or drop deformation is detected by the change of the internal optical path of a laser beam crossing perpendicular to the oscillation axis. Due to the closed geometry, a discrete spectrum of stationary oscillation frequencies (normal modes) is excited. The interferometric nature of the measurement and the resonant nature of the oscillation modes concur in allowing for high sensitivity, in the sub-nanometric region. We present a detailed description of the experimental setup and examples of applications of the technique to the study of both gas-liquid and liquid-liquid interfaces, either naked or with adsorbed surfactant monolayers, for bubbles and drops with diameter~1mm. In particular, the resonance frequencies and the width of the resonance peaks depend on the surface tension and the viscous dampening, respectively. We show that, by this new technique, properties of the interface can be accessed with confidence at the sub-nanometer scale, and surface phenomena, like the monolayer phase transition or the peculiarities of adsorption/desorption processes, can be unraveled in concentration regimes which are too low for existing methods.

Anomalous Behavior of Ultra-Low-Amplitude Capillary Waves. A Glimpse of the Viscoelastic Properties of Interfacial Water?

We investigate, both theoretically and by a differential interferometric technique, the behavior of large-wavelength capillary waves (of the order of 10-4 m) selectively excited at the surface of drops and bubbles with typical eigenfrequencies of the order of 102 Hz. The resonance peaks of gas bubbles or hydrocarbon drops in water (radius less than 1 mm) highlight anomalously small dissipation in the region of ultralow (sub-nanometric) oscillation amplitudes, reaching a plateau at higher amplitudes. This is in sharp contrast to the usual oscillating systems, where an anomalous behavior holds at large amplitudes alone. Dissipation is strongly dependent on the excited vibrational modes and, in spite of remarkable numerical differences, water-vapor and water-hydrocarbon interfaces exhibit the same overall trend. A phenomenological model was developed, based on the assumption that water possesses a threshold viscoelasticity, above which it behaves like a regular viscous fluid. The well-known Deborah number was then estimated within the anomalous region and found to lie in the range of viscoelastic fluids. In agreement with previous studies of nanohydrodynamics (e.g., atomic force microscopy measurements with sub-nanometric tip motions), the present one lends support to the idea that every self-aggregating fluid exhibits yield stress behavior, including classical Newtonian fluids like water. The essential requirement is that the applied perturbation lie below a critical threshold, above which viscous behavior is recovered. Our differential interferometric technique seems particularly suitable for this type of studies, as it allows measurement of long-wavelength capillary waves with sub-nanometric resolution on the oscillation amplitudes.

Oscillations of Bubble Shape Cause Anomalous Surfactant Diffusion: Experiments, Theory, and Simulations.

We investigate, both theoretically and experimentally, the role played by the oscillations of the cell membrane on the capture rate of substances freely diffusing around the cell. To obtain quantitative results, we propose and build up a reproducible and tunable biomimetic experimental model system to simulate the phenomenon of an oscillation-enhanced (or depressed) capture rate (chemoreception) of a diffusant. The main advantage compared to real biological systems is that the different oscillation parameters (type of deformation, frequencies, and amplitudes) can be finely tuned. The model system that we use is an anchored gas drop submitted to a diffusive flow of charged surfactants. When the surfactant meets the surface of the bubble, it is reversibly adsorbed. Bubble oscillations of the order of a few nanometers are selectively excited, and surfactant transport is accurately measured. The surfactant concentration past the oscillating bubbles was detected by conductivity measurements. The results highlight the role of surface oscillations on the diffusant capture rate. Particularly unexpected is the onset of intense overshoots during the adsorption process. The phenomenon is particularly relevant when the bubbles are exposed to intense forced oscillations near resonance.

Lipid-assisted protein transport: A diffusion-reaction model supported by kinetic experiments and molecular dynamics simulations.

The protein transport inside a cell is a complex phenomenon that goes through several difficult steps. The facilitated transport requires sophisticated machineries involving protein assemblies. In this work, we developed a diffusion-reaction model to simulate co-transport kinetics of proteins and lipids. We assume the following: (a) there is always a small lipid concentration of order of the Critical Micellar Concentration (CMC) in equilibrium with the membrane; (b) the binding of lipids to proteins modulates the hydrophobicity of the complexes and, therefore, their ability to interact and merge with the bilayer; and (c) some lipids leave the bilayer to replenish those bound to proteins. The model leads to a pair of integral equations for the time-evolution of the adsorbed proteins in the lipid bilayer. Relationships between transport kinetics, CMC, and lipid-protein binding constants were found. Under particular conditions, a perturbation analysis suggests the onset of kinks in the protein adsorption kinetics. To validate our model, we performed leakage measurements of vesicles composed by either high or low CMC lipids interacting with Islet Amyloid PolyPeptide (IAPP) and Aß (1-40) used as sample proteins. Since the lipid-protein complex stoichiometry is not easily accessible, molecular dynamics simulations were performed using monomeric IAPP interacting with an increasing number of phospholipids. Main results are the following: (a) 1:1 lipid-protein complexes generally show a faster insertion rate proportional to the complex hydrophobicity and inversely related to lipid CMC; (b) on increasing the number of bound lipids, the protein insertion rate decreases; and

Trapping of Sodium Dodecyl Sulfate at the Air-Water Interface of Oscillating Bubbles.

We report that at very low initial bulk concentrations, a couple of hundred times below the critical micellar concentration (CMC), anionic surfactant sodium dodecyl sulfate (SDS) adsorbed at the air-water interface of a gas bubble cannot be removed, on the time scale of the experiment (hours), when the surrounding solution is gently replaced by pure water. Extremely sensitive interferometric measurements of the resonance frequency of the bubble-forced oscillations give precise access to the concentration of the surfactant monolayer. The bulk-interface dynamic exchange of SDS molecules is shown to be inhibited below a concentration which we believe refers to a kind of gas-liquid phase transition of the surface monolayer. Above this threshold we recover the expected concentration-dependent desorption. The experimental observations are interpreted within simple energetic considerations supported by molecular dynamics (MD) calculations.

Resveratrol interferes with the aggregation of membrane-bound human-IAPP: a molecular dynamics study.

Amyloid aggregation of islet amyloid polypeptide (IAPP) in pancreatic tissues is a typical feature of type 2 diabetes mellitus. Resveratrol, a natural product extensively studied for its wide range of biological effects, has been shown to inhibit IAPP aggregation. However, the mechanism by which resveratrol inhibits IAPP aggregation is still far from complete elucidation. Now, an increasing knowledge of the mechanism of amyloid toxicity shifts the target of research towards the development of compounds which can prevent amyloid-mediated membrane damage rather than merely inhibit fiber formation. In this study we used all atom molecular dynamics to investigate the interaction of resveratrol with full-length human IAPP in a negatively charged membrane environment. Our results show that the presence of resveratrol induces the formation of secondary structures (sheets and helices) by inserting in a hydrophobic pocket between the interaction surface of two IAPP molecules in aqueous solution. On the other hand, resveratrol significantly perturbs the interaction of IAPP with negatively charged membranes by anchoring specific hydrophobic regions (23FGA25 and 32VGS34) of the peptide and forming a stable 1:2 IAPP:resveratrol complex at the water/membrane interphase.

Peptide-induced membrane curvature in edge-stabilized open bilayers: a theoretical and molecular dynamics study.

Peptide- or protein-induced curvatures of lipid membranes may be studied in molecular dynamics (MD) simulations. In these, membranes are usually modeled as infinitely extended bilayers by using periodic boundary conditions. However, the enforced periodicity results in an underestimation of the bending power of peptides, unless the patch size is much larger than the induced curvature radii. In this letter, we propose a novel approach to evaluate the bending power of a given distribution and/or density of peptides based on the use of flat open-edged lipid patches. To ensure long-lived metastable structures, the patch rim is stabilized in MD simulations by a local enrichment with short-chain lipids. By combining the theory of continuum elastic media with MD simulations, we prove that open-edged patches evolve from a planar state to a closed vesicle, with a transition rate that strongly depends on the concentration of lipid soluble peptides. For close-to-critical values for the patch size and edge energy, the response to even small changes in peptide concentration adopts a transition-like behavior (buckling instability). The usage of open-edged membrane patches amplifies the bending power of peptides, thereby enabling the analysis of the structural properties of membrane-peptide systems. We applied the presented method to investigate the curvature induced by aggregating ß -amyloid peptides, unraveling a strong sensitivity of membrane deformation to the peptide concentration.

Optimizing the crowding strategy: sugar-based ionic micelles in the dilute-to-condensed regime.

In the present study, we explore the effect of concentration on micelles made by different gangliosides, which are ionic biological glycolipids bearing multisugar headgroups with huge steric hindrance. Moreover, strong preferential interactions exist among like-conformer headgroups that can keep the ganglioside micelles in a trapped configuration. We extend the well-known ionic-amphiphiles paradigm, where local condensation and micelle crowding are matched by forming larger aggregates at increasing concentration. In fact, we force the balance between interparticle and intraparticle interactions while allowing for like conformers to modulate rebalancing. In the vast experimental framework, obtained by Small Angle X-ray scattering (SAXS) experiments, a theoretical model, accounting for a collective conformational transition of the bulky headgroups, is developed and successfully tested. It allows us to shed some light on the nature and coupling of the intermolecular forces involved in the interactions among glycolipid micelles. Energy minimization leads to complex behavior of the aggregation number on increasing concentration, fully consistent with the experimental landscape. From a biological perspective, this result could be reflected in the properties of ganglioside-enriched rafts on cell membranes, with a nonlinear structural response to approaching bodies such as charged proteins.

Hydrodynamic enhancement of the diffusion rate in the region between two fluctuating membranes in close opposition: a theoretical and computational study.

Periodic variation of the distance between two weakly adhering bodies gives rise to a huge tangential motions of the sandwiched solvent layer (squeezing flow). Oscillations either can be induced by an external applied field or can spontaneously arise from the coupling with the solvent heat bath. First we calculated by the Navier-Stokes equation the components of the fluid velocity near two oscillating juxtaposed plates. Then we evaluated the influence of plate oscillations on the transport properties of a trace diffusant dissolved at t = 0 in the outer medium for both deterministic and stochastic excitations. By employing both analytical (Fokker-Planck) and coarse-grained molecular dynamics (MD) simulations, we proved that the entry and migration rates of the diffusant sharply increases with the oscillation amplitudes. Enhancement was related to relevant parameters like oscillation frequency, fluid layer thickness, fluid viscosity, and temperature. An extension to the case of oscillating multistacked lamellae has been also made. Theoretical and MD results suggest a significant enhancement of the diffusant flux even in the worse situation of thermally excited small amplitude fluctuations. Excitation arising from other sources (e.g., microwave or ultrasound irradiation of solid-fluid layered systems) could have a dramatic effect on the transport phenomena. Possible implications to relevant biological problems have been discussed.

Simulation of polyethylene glycol and calcium-mediated membrane fusion.

We report on the mechanism of membrane fusion mediated by polyethylene glycol (PEG) and Ca(2+) by means of a coarse-grained molecular dynamics simulation approach. Our data provide a detailed view on the role of cations and polymer in modulating the interaction between negatively charged apposed membranes. The PEG chains cause a reduction of the inter-lamellar distance and cause an increase in concentration of divalent cations. When thermally driven fluctuations bring the membranes at close contact, a switch from cis to trans Ca(2+)-lipid complexes stabilizes a focal contact acting as a nucleation site for further expansion of the adhesion region. Flipping of lipid tails induces subsequent stalk formation. Together, our results provide a molecular explanation for the synergistic effect of Ca(2+) and PEG on membrane fusion.

Out of equilibrium divergence of dissipation in an oscillating bubble coated by surfactants.

We report measurements of the relaxation and resonance frequency of forced oscillating bubbles covered by a layer of surface-active molecules, the anionic surfactant sodium dodecyl sulfate (SDS). Less systematic investigations have been also carried out on neutral and cationic surfactants. A divergence of the viscous damping is observed at a very low bulk concentration. Subtle variations in the resonance peak are also measured. Bubble oscillations are driven by an electric field and measured with a sensitive interferometric technique. Results are interpreted with a model which takes care of the coupling between the dynamics of fluid surface oscillations and the properties of a surfactant monolayer in the vicinity of the phase transition from a gas-like distribution to a liquid-like assembly (the so-called gas-LE transition). Important charge effects are also considered. The basic assumptions of the model (cooperative adsorption of the surfactant at the air-water interface and coupling between the shape of the deformed surface and the local surfactant concentration) have been fully confirmed by extensive coarse-grained molecular dynamics simulations on model systems.

α-helical structures drive early stages of self-assembly of amyloidogenic amyloid polypeptide aggregate formation in membranes.

The human islet amyloid polypeptide (hIAPP) is the primary component in the toxic islet amyloid deposits in type-2 diabetes. hIAPP self-assembles to aggregates that permeabilize membranes and constitutes amyloid plaques. Uncovering the mechanisms of amyloid self-assembly is the key to understanding amyloid toxicity and treatment. Although structurally similar, hIAPP's rat counterpart, the rat islet amyloid polypeptide (rIAPP), is non-toxic. It has been a puzzle why these peptides behave so differently. We combined multiscale modelling and theory to explain the drastically different dynamics of hIAPP and rIAPP: The differences stem from electrostatic dipolar interactions. hIAPP forms pentameric aggregates with the hydrophobic residues facing the membrane core and stabilizing water-conducting pores. We give predictions for pore sizes, the number of hIAPP peptides, and aggregate morphology. We show the importance of curvature-induced stress at the early stages of hIAPP assembly and the α-helical structures over ß-sheets. This agrees with recent fluorescence spectroscopy experiments.

Anomalous viscosity effect in the early stages of the ion-assisted adhesion/fusion event between lipid bilayers: a theoretical and computational study.

The effect of viscosity on the encounter rate of two interacting membranes was investigated by combining a non-equilibrium Fokker-Planck model together with extensive Molecular Dynamics (MD) calculations. The encounter probability and stabilization of transient contact points represent the preliminary steps toward short-range adhesion and fusion of lipid leaflets. To strengthen our analytical model, we used a Coarse Grained MD method to follow the behavior of two charged palmitoyl oleoyl phosphatidylglycerol membranes embedded in a electrolyte-containing box at different viscosity regimes. Solvent friction was modulated by varying the concentration of a neutral, water-soluble polymer, polyethylene glycol, while contact points were stabilized by divalent ions that form bridges among juxtaposed membranes. While a naïve picture foresees a monotonous decrease of the membranes encounter rate with solvent viscosity, both the analytical model and MD simulations show a complex behavior. Under particular conditions, the encounter rate could exhibit a maximum at a critical viscosity value or for a critical concentration of bridging ions. These results seem to be confirmed by experimental observations taken from the literature.