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Beta-Amyloid Oligomer Formation in Physiological Solutions - a Study by Single-Molecule Fluorescence Resonance Energy Transfer
364a
Tuesday, February 14, 2017
1788-Pos Board B108 Probing the Mechanism of Glycosaminoglycan-Mediated Amyloid Assembly with a Non-Amyloidogenic Peptide Model Mathew Sebastiao1, Isabelle Marcotte1, Steve Bourgault1,2. 1 Chemistry, Universite´ du Que´bec a` Montre´al, Montreal, QC, Canada, 2 Quebec Network for Research on Protein Function, Structure, and Engineering (PROTEO), Quebec, QC, Canada. Over the last two decades, numerous studies have reported that glycosaminoglycans (GAGs) accelerate amyloid assembly of peptides and proteins whose aggregation is associated with amyloid-related diseases. GAGs are long, linear, unbranched polysaccharides that are abundant at the cell surface and in the extracellular matrix. These sulfated polysaccharides have been associated with virtually all amyloid extracts analyzed from patients afflicted with protein misfolding diseases. Strikingly, GAGs not only mediate amyloid assembly of aggregation-prone polypeptides but also the fibrilisation of numerous nonamyloidogenic protein. The exact mechanism by which GAGs favor peptide self-assembly, however, is still a matter of active debate. In this context, we used a non-amyloidogenic and highly soluble peptide, PACAP27, as a model to investigate this mechanism. PACAP27 is a 27-residue neurohormone that is non-pathogenic, stable in solution, and does not aggregate. In presence of low molecular weight heparin (LMWH) however, it readily forms amyloidlike fibrils. Interestingly, PACAP27 rapidly adopts an a-helical structure upon binding to sulfated GAGs and this conformation can be observed during the lag phase of the amyloid reaction. After a prolonged incubation, secondarystructural transitions into a b-sheet-rich conformation are observed. The amyloid nature of these assemblies were confirmed by atomic force microscopy, transmission electron microscopy, circular dichroism spectroscopy and thioflavin-T fluorescence. In order to study whether a-helical structures are essential to GAG-mediated amyloidogenic pathways, two synthetic variants with reduced helical folding propensity were designed. Conformationally restricted peptides were able to form amyloid fibrils despite increased resistance to a-helix formation. Lag phases from aggregation kinetics were observably reduced in both variants, suggesting that the formation of helical structures in the presence of GAGs is not an obligatory step in the mechanism of amyloid fibril formation. 1789-Pos Board B109 Probing the Binding of Ab Peptides to Lipid Bilayers Christopher Lockhart, Dmitri K. Klimov. School of Systems Biology, George Mason University, Manassas, VA, USA. Alzheimer’s disease pathogenesis has been related to the accumulation of Ab peptides on cellular membranes. In this work, we investigate the interactions of Ab peptides with zwitterionic DMPC bilayers in the absence or presence of calcium salt and with an anionic DMPS bilayer using isobaric-isothermal all-atom explicit-solvent replica-exchange molecular dynamics simulations. Importantly, these simulation systems probe the thermodynamic binding of Ab peptides to lipid bilayers and provide a detailed characterization of Ab conformational ensembles. We have found that both zwitterionic and anionic bilayers lead to a promotion of Ab helical conformations, although the anionic DMPS bilayer produces less helix than zwitterionic bilayers. When Ab binds to the anionic bilayer, it forms fewer intrapeptide contacts and is subsequently more expanded than when bound to zwitterionic DMPC bilayers. Ab penetrates into the bilayer hydrophobic core of zwitterionic bilayers in the absence or presence of calcium salt; however, in the latter system, binding is enhanced due in part to electrostatic cross-bridging of anionic amino acids, calcium ions, and negatively-charged lipid phosphate groups. Insertion of Ab into zwitterionic DMPC bilayers indents the bilayer structure, resulting in bilayer thinning and a lipid density depression. For comparison, the anionic DMPS bilayer prevents deep Ab insertion localizing amino acids to the bilayer surface via strong electrostatic interactions between charged amino acids and polar lipid headgroups. Not surprisingly, Ab binding to the DMPS bilayer results in less bilayer thinning than binding to DMPC bilayers. In all systems, the effect of Ab on the bilayer structure is limited to its immediate binding footprint. Ab peptides also fail to enhance the permeation of water through the bilayer. Collectively, these results provide a mechanistic explanation for the status of monomeric Ab peptides as an inert species. 1790-Pos Board B110 Beta-Amyloid Oligomer Formation in Physiological Solutions - a Study by Single-Molecule Fluorescence Resonance Energy Transfer Jun Han1, Erwen Mei2, Mei-Ping Kung3, Hank F. Kung3, Jian-Min Yuan4, Hai-Lung Dai1. 1 Department of Chemistry, Temple University, Philadelphia, PA, USA, 2 Regional Laser and Biotechnology Laboratories, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Radiology, University
of Pennsylvania, Philadelphia, PA, USA, 4Department of Physics, Drexel University, Philadelphia, PA, USA. Formation of oligomers of b-amyloid (Ab) peptides is a pivotal initial event associated with the pathogenicity of Alzheimer’s disease (AD). Using the single-molecule fluorescence resonance energy transfer (FRET) microscopy technique, we have detected the very beginning of the formation of Ab peptide oligomers in physiological solutions. The existence of oligomers is identified from the FRET between dyes individually bonded to the N-terminus of the 40-unit peptides. Several dimers and oligomers identified by the varying FRET efficiency have been detected. The dimer with the highest stability is ˚ between its two N-termini, in an characterized to have a distance of 43 A anti-parallel structure that is similar to that of the dimer unit (anti-parallel hairpin) that has been previously identified in the fibrils. 1791-Pos Board B111 Statistical Thermodynamic Modeling of Early Ab Oligomer Formation Nicholas P. van der Munnik1, Tao Wei2, Melissa A. Moss1, Mark J. Uline1. 1 Chemical Engineering, University of South Carolina, Columbia, SC, USA, 2 Chemical Engineering, Lamar University, Beaumont, TX, USA. Alzheimer’s disease (AD) is the most common neurodegenerative disease. A mounting body of research implicates amyloid-b (Ab) oligomers as an etiological factor in AD. However, the exact structure and stability of these species remains unclear. We have implemented an approach that uses Ab conformations obtained from replica exchange molecular dynamics (REMD) simulations in conjunction with a statistical thermodynamic model to derive thermodynamic information on Ab oligomer formation. Fully atomistic REMD simulations of Ab were performed using Gromacs, the Charmm 36 force field and the TIP3P water model. An Ab monomer was simulated using REMD at two different ionic strengths. Additionally, an Ab dimer was simulated at room temperature and physiological ionic strength. These simulations were used to derive an ensemble of conformations for both monomers in isolation and during the binding process. A self-consistent field theory has been developed to model the thermodynamics of interacting Ab molecules. The theory consists of formulating the appropriate thermodynamic potential for Ab molecules in a bath of solvent and ionic species in terms of the energy and entropy associated with all species. In this study, the centers of mass of three Ab monomers were assigned to positions in space, and the model was used to calculate the properties of equilibrium under those constraints. This procedure was repeated over a range of relative positions to elucidate the free energy landscape of the trimer formation process. This theory provides a rich description of the molecular organization and physics that guide Ab interactions that cannot be determined experimentally. The unique flexibility of this theoretical framework to treat solution conditions and systems of arbitrary geometries makes this a promising method to both further fundamental understanding of Ab oligomerization and serve as an inhibitor design tool. 1792-Pos Board B112 Strain Specific Propagation of an Amyloid Beta Oligomer in Alzheimer Disease Vijay Rangachari. University of Southern Mississippi, Hattiesburg, MS, USA. Low-molecular weight oligomers of amyloid-b (Ab) have now been understood as the primary neurotoxic agents responsible for synaptic dysfunction and neuronal abnormalities in Alzheimer disease (AD) patients. Alongside cellular toxicity, aggregates of Ab are also involved in proliferation and spreading of toxicity, the mechanism of which remains unclear, especially the role of oligomers in such a process. Emerging pathological evidence indicate prion-like propagation among Ab aggregates, which may govern their ability to proliferate. Furthermore, polymorphism observed within the aggregation end products of fibrils are known to arise due to microstructural differences among the oligomers. Diversity in aggregate morphology correlates with the observed phenotypes in AD, cementing the idea that conformational strains of oligomers could be significant in phenotypic outcomes. Therefore, it is imperative to determine the ability of oligomeric strains to faithfully propagate their structure. We have identified a 12-mer of Ab42 called, large fatty acidderived oligomer (LFAOs), which replicate upon interacting with monomers to generate quantitative amount of LFAOs. Replication is efficient at low concentrations (< 10 mM), where LFAOs is predominantly a 12mer. At higher concentrations, LFAOs are predominantly 12-24mers that are less efficient for replication. However, 12-24mers undergo propagation towards morphologically distinct fibrils, one that is made of discrete LFAO building blocks. These results establish that strain-specific propagation of oligomeric Ab takes places in two distinct steps of amplification and propagation in low and high concentrations of the seed, respectively. These data have opened doors to understanding underlying mechanisms of proliferation as well as strain-specific phenotypic outcomes in AD.
Tuesday, February 14, 2017
1788-Pos Board B108 Probing the Mechanism of Glycosaminoglycan-Mediated Amyloid Assembly with a Non-Amyloidogenic Peptide Model Mathew Sebastiao1, Isabelle Marcotte1, Steve Bourgault1,2. 1 Chemistry, Universite´ du Que´bec a` Montre´al, Montreal, QC, Canada, 2 Quebec Network for Research on Protein Function, Structure, and Engineering (PROTEO), Quebec, QC, Canada. Over the last two decades, numerous studies have reported that glycosaminoglycans (GAGs) accelerate amyloid assembly of peptides and proteins whose aggregation is associated with amyloid-related diseases. GAGs are long, linear, unbranched polysaccharides that are abundant at the cell surface and in the extracellular matrix. These sulfated polysaccharides have been associated with virtually all amyloid extracts analyzed from patients afflicted with protein misfolding diseases. Strikingly, GAGs not only mediate amyloid assembly of aggregation-prone polypeptides but also the fibrilisation of numerous nonamyloidogenic protein. The exact mechanism by which GAGs favor peptide self-assembly, however, is still a matter of active debate. In this context, we used a non-amyloidogenic and highly soluble peptide, PACAP27, as a model to investigate this mechanism. PACAP27 is a 27-residue neurohormone that is non-pathogenic, stable in solution, and does not aggregate. In presence of low molecular weight heparin (LMWH) however, it readily forms amyloidlike fibrils. Interestingly, PACAP27 rapidly adopts an a-helical structure upon binding to sulfated GAGs and this conformation can be observed during the lag phase of the amyloid reaction. After a prolonged incubation, secondarystructural transitions into a b-sheet-rich conformation are observed. The amyloid nature of these assemblies were confirmed by atomic force microscopy, transmission electron microscopy, circular dichroism spectroscopy and thioflavin-T fluorescence. In order to study whether a-helical structures are essential to GAG-mediated amyloidogenic pathways, two synthetic variants with reduced helical folding propensity were designed. Conformationally restricted peptides were able to form amyloid fibrils despite increased resistance to a-helix formation. Lag phases from aggregation kinetics were observably reduced in both variants, suggesting that the formation of helical structures in the presence of GAGs is not an obligatory step in the mechanism of amyloid fibril formation. 1789-Pos Board B109 Probing the Binding of Ab Peptides to Lipid Bilayers Christopher Lockhart, Dmitri K. Klimov. School of Systems Biology, George Mason University, Manassas, VA, USA. Alzheimer’s disease pathogenesis has been related to the accumulation of Ab peptides on cellular membranes. In this work, we investigate the interactions of Ab peptides with zwitterionic DMPC bilayers in the absence or presence of calcium salt and with an anionic DMPS bilayer using isobaric-isothermal all-atom explicit-solvent replica-exchange molecular dynamics simulations. Importantly, these simulation systems probe the thermodynamic binding of Ab peptides to lipid bilayers and provide a detailed characterization of Ab conformational ensembles. We have found that both zwitterionic and anionic bilayers lead to a promotion of Ab helical conformations, although the anionic DMPS bilayer produces less helix than zwitterionic bilayers. When Ab binds to the anionic bilayer, it forms fewer intrapeptide contacts and is subsequently more expanded than when bound to zwitterionic DMPC bilayers. Ab penetrates into the bilayer hydrophobic core of zwitterionic bilayers in the absence or presence of calcium salt; however, in the latter system, binding is enhanced due in part to electrostatic cross-bridging of anionic amino acids, calcium ions, and negatively-charged lipid phosphate groups. Insertion of Ab into zwitterionic DMPC bilayers indents the bilayer structure, resulting in bilayer thinning and a lipid density depression. For comparison, the anionic DMPS bilayer prevents deep Ab insertion localizing amino acids to the bilayer surface via strong electrostatic interactions between charged amino acids and polar lipid headgroups. Not surprisingly, Ab binding to the DMPS bilayer results in less bilayer thinning than binding to DMPC bilayers. In all systems, the effect of Ab on the bilayer structure is limited to its immediate binding footprint. Ab peptides also fail to enhance the permeation of water through the bilayer. Collectively, these results provide a mechanistic explanation for the status of monomeric Ab peptides as an inert species. 1790-Pos Board B110 Beta-Amyloid Oligomer Formation in Physiological Solutions - a Study by Single-Molecule Fluorescence Resonance Energy Transfer Jun Han1, Erwen Mei2, Mei-Ping Kung3, Hank F. Kung3, Jian-Min Yuan4, Hai-Lung Dai1. 1 Department of Chemistry, Temple University, Philadelphia, PA, USA, 2 Regional Laser and Biotechnology Laboratories, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Radiology, University
of Pennsylvania, Philadelphia, PA, USA, 4Department of Physics, Drexel University, Philadelphia, PA, USA. Formation of oligomers of b-amyloid (Ab) peptides is a pivotal initial event associated with the pathogenicity of Alzheimer’s disease (AD). Using the single-molecule fluorescence resonance energy transfer (FRET) microscopy technique, we have detected the very beginning of the formation of Ab peptide oligomers in physiological solutions. The existence of oligomers is identified from the FRET between dyes individually bonded to the N-terminus of the 40-unit peptides. Several dimers and oligomers identified by the varying FRET efficiency have been detected. The dimer with the highest stability is ˚ between its two N-termini, in an characterized to have a distance of 43 A anti-parallel structure that is similar to that of the dimer unit (anti-parallel hairpin) that has been previously identified in the fibrils. 1791-Pos Board B111 Statistical Thermodynamic Modeling of Early Ab Oligomer Formation Nicholas P. van der Munnik1, Tao Wei2, Melissa A. Moss1, Mark J. Uline1. 1 Chemical Engineering, University of South Carolina, Columbia, SC, USA, 2 Chemical Engineering, Lamar University, Beaumont, TX, USA. Alzheimer’s disease (AD) is the most common neurodegenerative disease. A mounting body of research implicates amyloid-b (Ab) oligomers as an etiological factor in AD. However, the exact structure and stability of these species remains unclear. We have implemented an approach that uses Ab conformations obtained from replica exchange molecular dynamics (REMD) simulations in conjunction with a statistical thermodynamic model to derive thermodynamic information on Ab oligomer formation. Fully atomistic REMD simulations of Ab were performed using Gromacs, the Charmm 36 force field and the TIP3P water model. An Ab monomer was simulated using REMD at two different ionic strengths. Additionally, an Ab dimer was simulated at room temperature and physiological ionic strength. These simulations were used to derive an ensemble of conformations for both monomers in isolation and during the binding process. A self-consistent field theory has been developed to model the thermodynamics of interacting Ab molecules. The theory consists of formulating the appropriate thermodynamic potential for Ab molecules in a bath of solvent and ionic species in terms of the energy and entropy associated with all species. In this study, the centers of mass of three Ab monomers were assigned to positions in space, and the model was used to calculate the properties of equilibrium under those constraints. This procedure was repeated over a range of relative positions to elucidate the free energy landscape of the trimer formation process. This theory provides a rich description of the molecular organization and physics that guide Ab interactions that cannot be determined experimentally. The unique flexibility of this theoretical framework to treat solution conditions and systems of arbitrary geometries makes this a promising method to both further fundamental understanding of Ab oligomerization and serve as an inhibitor design tool. 1792-Pos Board B112 Strain Specific Propagation of an Amyloid Beta Oligomer in Alzheimer Disease Vijay Rangachari. University of Southern Mississippi, Hattiesburg, MS, USA. Low-molecular weight oligomers of amyloid-b (Ab) have now been understood as the primary neurotoxic agents responsible for synaptic dysfunction and neuronal abnormalities in Alzheimer disease (AD) patients. Alongside cellular toxicity, aggregates of Ab are also involved in proliferation and spreading of toxicity, the mechanism of which remains unclear, especially the role of oligomers in such a process. Emerging pathological evidence indicate prion-like propagation among Ab aggregates, which may govern their ability to proliferate. Furthermore, polymorphism observed within the aggregation end products of fibrils are known to arise due to microstructural differences among the oligomers. Diversity in aggregate morphology correlates with the observed phenotypes in AD, cementing the idea that conformational strains of oligomers could be significant in phenotypic outcomes. Therefore, it is imperative to determine the ability of oligomeric strains to faithfully propagate their structure. We have identified a 12-mer of Ab42 called, large fatty acidderived oligomer (LFAOs), which replicate upon interacting with monomers to generate quantitative amount of LFAOs. Replication is efficient at low concentrations (< 10 mM), where LFAOs is predominantly a 12mer. At higher concentrations, LFAOs are predominantly 12-24mers that are less efficient for replication. However, 12-24mers undergo propagation towards morphologically distinct fibrils, one that is made of discrete LFAO building blocks. These results establish that strain-specific propagation of oligomeric Ab takes places in two distinct steps of amplification and propagation in low and high concentrations of the seed, respectively. These data have opened doors to understanding underlying mechanisms of proliferation as well as strain-specific phenotypic outcomes in AD.