Calcium Enhances Binding of Aβ Monomer to DMPC Lipid Bilayer

Using isobaric-isothermal replica-exchange molecular dynamics and the all-atom explicit-solvent model, we studied the equilibrium binding of Aβ monomers to a zwitterionic dimyristoylphosphatidylcholine (DMPC) bilayer coincubated with calcium ions. Using our previous replica-exchange molecular dynamics calcium-free simulations as a control, we reached three conclusions. First, calcium ions change the tertiary structure of the bound Aβ monomer by destabilizing several long-range intrapeptide interactions, particularly the salt bridge Asp²³-Lys²⁸. Second, calcium strengthens Aβ peptide binding to the DMPC bilayer by enhancing electrostatic interactions between charged amino acids and lipid polar headgroups. As a result, Aβ monomer penetrates deeper into the bilayer, making disorder in proximal lipids and bilayer thinning more pronounced. Third, because calcium ions demonstrate strong affinity to negatively charged amino acids, a considerable influx of calcium into the area proximal to the bound Aβ monomer is observed. Consequently, the localizations of negatively charged amino acids and calcium ions in the Aβ binding footprint overlap. Based on our data, we propose a mechanism by which calcium ions strengthen Aβ-bilayer interactions. This mechanism involves two factors: 1) calcium ions make the DMPC bilayer partially cationic and thus attractive to the anionic Aβ peptide; and 2) destabilization of the Asp²³-Lys²⁸ salt bridge makes Lys²⁸ available for interactions with the bilayer. Finally, we conclude that a single Aβ monomer does not promote permeation of calcium ions through the zwitterionic bilayer.     

Insertion into lipid bilayer of truncated pHLIP® peptide

The investigation of pH-dependent membrane-associated folding has both fundamental interest and practical applications for targeting of acidic tumors and specific delivery of therapeutic molecules across membrane of cancer cells. We and others investigated molecular mechanism and medical uses of class of water soluble membrane peptides, pH (Low) Insertion Peptides (pHLIP^® peptides). Here we employed optical spectroscopy methods to study interactions of the truncated pHLIP^® peptide (Short pHLIP^®) with lipid bilayer of membrane. Tryptophan fluorescence, CD and OCD data indicate on pH-triggered formation of transmembrane helical structure. Dual quenching and FRET assays demonstrated that Short pHLIP^® peptide spans lipid bilayer of membrane similar to Long pHLIP^® peptides. Truncated pHLIP^® peptides with multiple charged and protonatable residues in their sequences potentially can make these peptides to be less hydrophobic compared to Long pHLIP^® peptides, and might have utility in tumor imaging, and potentially, in pH-regulated cytoplasmic delivery of moderately hydrophobic drugs.     

Alzheimer Aβ peptide interactions with lipid membranes

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Binding to the lipid monolayer induces conformational transition in Aβ monomer

Using implicit solvent atomistic model and replica exchange molecular dynamics, we study binding of Aβ monomer to zwitterionic dimyristoylphosphatidylcholine (DMPC) lipid monolayer. Our results suggest that Aβ binding to the monolayer is governed primarily by positively charged and aromatic amino acids. Lysine residues tend to interact with surface choline and phosphorous lipid groups, whereas aromatic amino acids penetrate deeper into the monolayer, reaching its hydrophobic core. We show that binding to the DMPC monolayer causes a dramatic conformational transition in Aβ monomer, resulting in chain extension, loss of intrapeptide interactions, and formation of β-structure. This conformational transition is far more significant than that occurring during the initial stages of aggregation in water. We also found that Aβ binding perturbs surface ordering of lipids interacting with Aβ.     

Binding,folding and insertion of a β-hairpin peptide at a lipid bilayer surface: Influence of electrostatics and lipid tail packing

Antimicrobial peptides (AMPs) act as host defenses against microbial pathogens. Here we investigate the interactions of SVS-1 (KVKVKVKV^dP^lPTKVKVKVK), an engineered AMP and anti-cancer β-hairpin peptide, with lipid bilayers using spectroscopic studies and atomistic molecular dynamics simulations. In agreement with literature reports, simulation and experiment show preferential binding of SVS-1 peptides to anionic over neutral bilayers. Fluorescence and circular dichroism studies of a Trp-substituted SVS-1 analogue indicate, however, that it will bind to a zwitterionic DPPC bilayer under high-curvature conditions and folds into a hairpin. In bilayers formed from a 1:1 mixture of DPPC and anionic DPPG lipids, curvature and lipid fluidity are also observed to promote deeper insertion of the fluorescent peptide. Simulations using the CHARMM C36m force field offer complementary insight into timescales and mechanisms of folding and insertion. SVS-1 simulated at an anionic mixed POPC/POPG bilayer folded into a hairpin over a microsecond, the final stage in folding coinciding with the establishment of contact between the peptide's valine sidechains and the lipid tails through a “flip and dip” mechanism. Partial, transient folding and superficial bilayer contact are seen in simulation of the peptide at a zwitterionic POPC bilayer. Only when external surface tension is applied does the peptide establish lasting contact with the POPC bilayer. Our findings reveal the influence of disruption to lipid headgroup packing (via curvature or surface tension) on the pathway of binding and insertion, highlighting the collaborative effort of electrostatic and hydrophobic interactions on interaction of SVS-1 with lipid bilayers.     

Binding studies of truncated variants of the Aβ peptide to the V-domain of the RAGE receptor reveal Aβ residues responsible for binding

Alzheimer's disease (AD) symptoms correlate with the concentration of soluble, although not necessarily monomeric forms of Aβ peptide in the brain parenchyma. The RAGE receptor has been implicated as the protein responsible for active transport of Aβ from blood circulation to the brain. In murine models of AD, inhibition of the Aβ:RAGE interaction decreases the levels of Aβ in the brain. Inhibition of the Aβ:RAGE interaction would be a promising alternative for the therapy of AD. Rational design of an Aβ:RAGE interaction blocker requires detailed knowledge of the structure of the complex. However, the binding domain of RAGE is natively unfolded in physiological conditions, which severely hampers the application of classic methods of protein structure analysis to the design of an antagonist. Here, alternative methods are used to characterize the structural properties of the RAGE-ligand binding domain and to monitor the binding of a series of truncated variants of Aβ. Using intrinsic RAGE tryptophan fluorescence and mass spectrometry of non-covalent protein-ligand complexes we have identified shorter versions of Aβ that bind to the RAGE V-domain. We have also shown in cell culture experiments that a selected shortened version of Aβ effectively inhibits full-length Aβ, RAGE-mediated, cell uptake. Thus, a truncated version of Aβ capable of blocking its receptor-mediated internalization was established, revealing the binding code and providing the lead compound in the process of drug design.     

Structure of the antimicrobial β-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation

All atom molecular dynamics simulations of the 18-residue β-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH₂) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of the peptide in a membrane environment (previously solved only in solution [R.L. Fahrner, T. Dieckmann, S.S.L. Harwig, R.I. Lehrer, D. Eisenberg, J. Feigon, Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes. Chemistry and Biology, 3 (1996) 543-550]), and to delineate specific peptide-membrane interactions which are responsible for the peptide's membrane binding properties. A novel, previously unknown, “kick” shaped conformation of the peptide was detected, where a bend at the C-terminal β-strand of the peptide caused the peptide backbone at residues 16-18 to extend perpendicular to the β-hairpin plane. This bend was driven by a highly persistent hydrogen-bond between the polar peptide side-chain of TYR7 and the unshielded backbone carbonyl oxygen atom of GLY17. The H-bond formation relieves the unfavorable free energy of insertion of polar groups into the hydrophobic membrane core. PG-1 was anchored to the membrane by strong electrostatic binding of the protonated N-terminus of the peptide to the lipid head group phosphate anions. The orientation of the peptide in the membrane, and its influence on bilayer structural and dynamic properties are in excellent agreement with solid state NMR measurements [S. Yamaguchi, T. Hong, A. Waring, R.I. Lehrer, M. Hong, Solid-State NMR Investigations of Peptide-Lipid Interaction and Orientation of a b-Sheet Antimicrobial Peptide, Protegrin, Biochemistry, 41 (2002) 9852-9862]. Importantly, two simulations which started from different initial orientations of the peptide converged to the same final equilibrium orientation of the peptide relative to the bilayer. The kick-shaped conformation was observed only in one of the two simulations.     

A model for the lateral diffusion of “stiff” chains in a lipid bilayer

We present random walk models for the diffusive motion of lipid probe molecules in a lipid bilayer and calculate the diffusion constants for probes spanning the entire bilayer and for probes extending through one lipid layer only. The stiffness of such molecules can explain the observed value of 2/3 for the ratio of these diffusion constants.     

Oligomeric amyloid-β peptide affects the expression of genes involved in steroid and lipid metabolism in primary neurons

Amyloid-β peptide (Aβ) is the principal component of plaques in the brains of patients with Alzheimer's disease (AD), and the most toxic form of Aβ may be as soluble oligomers. We report here the results of a microarray study of gene expression profiles in primary mouse cortical neurons in response to oligomeric Aβ(1-42). A major and unexpected finding was the down-regulation of genes involved in the biosynthesis of cholesterol and other steroids and lipids (such as Fdft1, Fdps, Idi1, Ldr, Mvd, Mvk, Nsdhl, Sc4mol), the expression of which was verified by quantitative real-time RT-PCR (qPCR). The ATP-binding cassette gene Abca1, which has a major role in cholesterol transport in brain and other tissues and has been genetically linked to AD, was notably up-regulated. The possible involvement of cholesterol and other lipids in Aβ synthesis and action in Alzheimer's disease has been studied and debated extensively but remains unresolved. These new data suggest that Aβ may influence steroid and lipid metabolism in neurons via multiple gene-expression changes.     

Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO₂ and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P₂ (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P₂ and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.     

Interactions of amyloid-β peptides on lipid bilayer studied by single molecule imaging and tracking

The amyloid-β peptides (Aβ40 and Aβ42) feature prominently in the synaptic dysfunction and neuronal loss associated with Alzheimer's disease (AD). This has been proposed to be due either to interactions between Aβ and cell surface receptors affecting cell signaling, or to the formation of calcium-permeable channels in the membrane that disrupt calcium homeostasis. In both mechanisms the cell membrane is the primary cellular structure with which Aβ interacts. Aβ concentrations in human bodily fluids are very low (pM-nM) rendering studies of the size, composition, cellular binding sites and mechanism of action of the oligomers formed in vivo very challenging. Most studies, therefore, have utilized Aβ oligomers prepared at micromolar peptide concentrations, where Aβ forms oligomeric species which possess easily observable cell toxicity. Such toxicity has not been observed when nM concentrations of peptide are used in the experiment highlighting the importance of employing physiologically relevant peptide concentrations for the results to be of biological significance. In this paper single-molecule microscopy was used to monitor Aβ oligomer formation and diffusion on a supported lipid bilayer at nanomolar peptide concentrations. Aβ monomers, the dominant species in solution, tightly associate with the membrane and are highly mobile whereas trimers and higher-order oligomers are largely immobile. Aβ dimers exist in a mixture of mobile and immobile states. Oligomer growth on the membrane is more rapid for Aβ40 than for the more amyloidogenic Aβ42 but is largely inhibited for a 1:1 Aβ40:Aβ42 mixture. The mechanism underlying these Aβ40-Aβ42 interactions may feature in Alzheimer's pathology.     

Modeling analysis of the lipid bilayer–cytoskeleton coupling in erythrocyte membrane

Studies of thermal fluctuations in discocytes, echinocytes, and spherocytes suggest that the coupling between lipid bilayer and cytoskeleton can affect viscoelastic behavior of single erythrocyte membranes. To test this hypothesis, we developed a 3D constitutive model describing viscoelastic behavior of erythrocyte membranes, at long relaxation times \(t \in [0.20\,\mathrm {s}, 1.05\,\mathrm {s}]\) and short relaxation times \(t \in [0.03\,\mathrm {s}, 0.20\,\mathrm {s}]\) . The model was constructed using combination of spring and spring pot rheological elements arranged in parallel. The rearrangement of cytoskeleton induced by changing the bending state of lipid bilayer was described by a modified Eyring model. The model predictions point to an anomalous nature of energy dissipation and an ordered harmonic nature of the coupling mechanism described by a series of fractional derivatives of the order n \(\alpha \) (where \( n \in [- 1, 2]\) ). As a result, the stress generated within the lipid bilayer is related to the rate of change of the irreversible stress within the cytoskeleton.     

Molecular interactions of Alzheimer's Aβ protofilaments with lipid membranes

Amyloid fibrils and peptide oligomers play central roles in the pathology of Alzheimer's disease, type 2 diabetes, Parkinson's disease, Huntington's disease, and prion-related disease. Here, we investigate the molecular interactions between preformed amyloid β (Aβ) molecular protofilaments and lipid bilayer membranes, in the presence of explicit water molecules, using computational models and all-atom molecular dynamics. These interactions play an important role in the stability and function of both Aβ fibrils and the adjacent cellular membrane. Taking advantage of the symmetry-related and directional properties of the protofilaments, we build models that cover several relative protofilament-membrane orientations. Our molecular dynamics simulations reveal the relative contributions of different structural elements to the dynamics and stability of Aβ protofilament segments near membranes, and the first steps in the mechanism of fibril-membrane interactions. During this process, we observe a significant alteration of the side-chain contact pattern in protofilaments, although a fraction of the characteristic β-sheet content is preserved. As a major driving force, we identify the electrostatic interactions between Aβ charged side chains, including E22, D23, and K28, and lipid headgroups. Together with hydrogen bonding with atoms from lipid headgroups, these interactions can facilitate the penetration of hydrophobic C-terminal amino acids through the lipid headgroup region, which can finally lead both to further loss of the initial fibril structure and to local membrane-thinning effects. Our results may guide new experiments that could test the extent to which the structural features of water-formed amyloid fibrils are preserved, lost, or reshaped by membrane-mediated interactions.     

Point mutations in Aβ result in the formation of distinct polymorphic aggregates in the presence of lipid bilayers

A hallmark of Alzheimer's disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.     

The effect of variations in phospholipid and sterol structure on the nature of lipid–sterol interactions in lipid bilayer model membranes

This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure.     

Differentiation of subnucleus-sized oligomers and nucleation-competent assemblies of the Aβ peptide

A significant feature of Alzheimer’s disease is the formation of amyloid deposits in the brain consisting mainly of misfolded derivatives of proteolytic cleavage products of the amyloid precursor protein amyloid-β (Aβ) peptide. While high-resolution structures already exist for both the monomer and the amyloid fibril of the Aβ peptide, the mechanism of amyloid formation itself still defies precise characterization. In this study, low and high molecular weight oligomers (LMWOs and HMWOs) were identified by sedimentation velocity analysis, and for the first time, the temporal evolution of oligomer size distributions was correlated with the kinetics of amyloid formation as determined by thioflavin T-binding studies. LMWOs of subnucleus size contain fewer than seven monomer units and exist alongside a heterogeneous group of HMWOs with 20–160 monomer units that represent potential centers of nucleus formation due to high local monomer concentrations. These HMWOs already have slightly increased β-strand content and appear structurally similar regardless of size, as shown by examination with a range of fluorescent dyes. Once fibril nuclei are formed, the monomer concentration begins to decrease, followed by a decrease in oligomer concentration, starting with LMWOs, which are the least stable species. The observed behavior classifies the two LMWOs as off pathway. In contrast, we consider HMWOs to be on-pathway, prefibrillar intermediates, representing structures in which nucleated conformational conversion is facilitated by high local concentrations. Aβ40 and Aβ42 M35^ox take much longer to form nuclei and enter the growth phase than Aβ42 under identical reaction conditions, presumably because both the size and the concentration of HMWOs formed are much smaller.     

Alzheimer Aβ peptide interactions with lipid membranes: Fibrils,oligomers and polymorphic amyloid channels

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Binding of small alcohols to a lipid bilayer membrane: does the partitioning coefficient express the net affinity?

The total vapor pressures at 26 degreesC of binary (water-alcohol) and ternary (water-alcohol-vesicle) systems were measured for six short chain alcohols. The vesicles were unilamellar dipalmitoyl phosphatidylcholine (DMPC). The data was used to evaluate the effect of vesicles on the chemical potential of alcohols expressed as the preferential binding parameter of the alcohol-lipid interaction, gamma23. This quantity is a thermodynamic (model-free) measure of the net strength of membrane-alcohol interactions. For the smaller investigated alcohols (methanol, ethanol and 1-propanol) gamma23 was negative. This is indicative of so-called preferential hydration, a condition where the affinity of the membrane for water is higher than the affinity for the alcohol. For the longer alcohols (1-butanol, 1-pentanol, 1-hexanol) gamma23 was positive and increasing with increasing chain length. This demonstrates preferential binding, i.e. enrichment of alcohol in the membrane and a concomitant depletion of the solute in the aqueous bulk. The measured values of gamma23 were compared to the number of alcohol-membrane contacts specified by partitioning coefficients from the literature. It was found that for the small alcohols the number of alcohol-membrane contacts is much larger than the number of preferentially bound solutes. This discrepancy, which is theoretically expected in cases of very weak binding, becomes less pronounced with increasing alcohol chain length, and when the partitioning coefficient exceeds approximately 3 on the molal scale (10(2) in mole fraction units) it vanishes. Based on this, relationships between structural and thermodynamic interpretations of membrane partitioning are discussed.