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X-Ray Digital Aggregated Dynamics of Intrinsically Disordered Proteins
Wednesday, March 2, 2016
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2726-Pos Board B103 Self-Assembly of Full-Size Amyloid Beta 40 Proteins in Dimers Mohtadin Hashemi, Yuliang Zhang, Zhengjian Lv, Yuri L. Lyubchenko. Dept. of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA. Even with growing evidence for the involvement of protein oligomeric nanoassemblies in the development of protein misfolding diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) among others, very limited knowledge exists regarding the molecular mechanisms underlying these processes. This is primarily because the protein aggregates are stabilized by weak interactions that are typically transient in nature and difficult to measure. Therefore, only mixtures of aggregates with different morphologies have been studied thus far; in turn, it is unknown how the aggregation process is initiated and how the growth of oligomers progresses. On the other hand, computational approaches can provide detailed descriptions of how monomers bind to the segments of fibrils. However, these powerful simulations, which provide the structure and dynamics of aggregates at the atomic level, require knowledge of the initial nanoassembly structure; in turn, computational analysis primarily models the elongation process of fibrils. Thus, available computational approaches can be applied to modeling the assembly of oligomers if the structure of the oligomer is known. Herein, we have conducted in silico simulations of Ab40 monomer assembly in dimers. The simulated structures were validated by comparing Monte Carlo pulling simulations with experimental data from single molecule AFM force spectroscopy enabling us to identify dimers structures rupture of which were in good agreement with the experimental results. One important observation from our long time-scale MD simulations is that the dimer does not have the extended beta-structures observed in fibrils. Rather, it remains largely unstructured and is stabilized by hydrophobic interaction and local hydrogen bonds. The results suggest that the Ab40 aggregation is a complex process in which the monomer structure depends on the aggregate size.
Institute, University of California, Santa Barbara, Santa Barbara, CA, USA, Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, CA, USA. Dysfunction of Tau, an intrinsically disordered protein localized to neuronal axons, is associated with many neurodegenerative diseases (‘‘Tauopathies’’) including FTDP-17, Alzheimer’s, and, more recently, chronic traumatic encephalopathy. However, that association is not fully understood, as the accepted, physiological functions of Tau are limited to stabilizing individual microtubules and mediating microtubule bundles. The latter conclusion is largely derived from seminal studies where transfected Tau cDNA prompted cells in culture to grow neurite-like processes with hexagonally-ordered microtubule arrays. However, ex vivo TEM of the axon shows only linear bundles of microtubules with no long-range positional ordering. Additionally, prior cell-free reconstitutions of microtubules with Tau exhibited either no bundles or bundles of dramatically different morphologies. The use of taxol in these reconstitutions further complicated matters; while making experiments tractable (stabilizing microtubules from depolymerization), taxol has been shown to weakly compete with Tau and affect microtubule assembly dynamics. Herein, we reconcile this conflicting behavior by reporting on the cell-free and taxolfree reconstitution of microtubules co-assembled with Tau at near-physiological conditions, recapitulating both hexagonally-symmetric and linear microtubule bundles. Small-angle X-ray scattering and TEM was used to obtain angstromresolution ensemble-averaged structural information and local morphology, respectively, of Tau-mediated MT bundles with large inter-MT spacings. Through an examination of truncated Tau and the force-response of microtubule bundles using osmotic depletants, we present a novel mechanism for long-ranged attractions between microtubules that is contingent on both the polyampholytic and intrinsically disordered nature of Tau. This proposed mechanism has heretofore been unseen in biology and would be significantly affected by Tau posttranslational modifications associated with dysfunction and neurodegeneration.
2727-Pos Board B104 X-Ray Digital Aggregated Dynamics of Intrinsically Disordered Proteins Naruki Hara1, Yufuku Matsushita1, Keigo Ikezaki1, Hiroshi Sekiguchi2, Naoya Fukui3, Yasushi Kawata3, Yuji C. Sasaki1,2. 1 The University of Tokyo, Chiba, Japan, 2JASRI/SPring-8, Hyogo, Japan, 3 Tottori University, Tottori, Japan. Parkinson’s Disease (PD) and Alzheimer Disease (AD) is one of the neurodegenerative pathology. These diseases are occurred by anomalous aggregations of causative proteins. Alpha-synuclein (Syn) is PD’s causative proteins and Tau is AD’s ones. But these protein’s aggregation process haven’t been clear. Additionally, these proteins are one of the famous intrinsically disorder proteins (IDP). Until now, it is difficult to understand aggregation process from static structural information of IDP. Therefore, in this study, we observed that characteristic of single molecule structural fluctuations which occurred anomalous aggregation. Furthermore, we observed dynamical digital aggregation process (from monomer to dimer or trimer). In order to understand single molecular dynamics which occurred aggregation, we measured single molecule structural fluctuations of Syn’s wild type (WT) and mutants (E46K, A53T). And, we observed digital aggregations of Tau proteins for the purpose of detecting dynamical process. At these experiments, we used Diffracted X-ray Tracking (DXT). In order to measure movements of the specific binding sites of proteins, DXT monitor X-ray diffraction spots from labeled gold nanocrystal. DXT experiments used the energy of quasi-white x-rays (energy peak-width of 2%, 10-20 keV, BL40XU, SPring-8), for that reason DXT has high time-resolution (0.1ms/frame) and high super-precision (0.1nm scale). As a result from Syn’s DXT data, The motion’s histograms in WT has simple single Gaussian distribution. However, these in other Syn’s mutants which occurred anomalous aggregation have two Gaussian distributions. From DXT results, we confirmed that mutant’s structural fluctuation is more rigid than that of WT. In the other hand, Tau dimer is very more flexible than that of monomer and trimer. The motion histograms show that there are very different process between dimerization and trimerization in the digital aggregation observations of Tau proteins.
2729-Pos Board B106 Ab Fibrils Act as Aqueous Pores: A Molecular Dynamics Study Sachin R. Natesh1, Stephen C. Meredith2, Tobin R. Sosnick3, Karl F. Freed4, Esmael J. Haddadian5. 1 Physical Sciences Collegiate Division, University of Chicago, Chicago, IL, USA, 2Dept. of Pathology and Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 3Dept. of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 4Department of Chemistry and James Frank Institute, University of Chicago, Chicago, IL, USA, 5Biological Sciences Collegiate Division, University of Chicago, Chicago, IL, USA. Alzheimer’s Disease (AD) is an increasingly prevalent neurodegenerative disease. Aggregation of Ab peptides is important in etiology of AD. We ran all-atom, explicit water molecular dynamics simulations starting from three experimentally available NMR structures of Ab fibrils, containing either 6 or an infinite number of layers. Infinite fibrils were constructed using periodic images. The NMR structures have either 2-fold (2LMN) or 3-fold rotational symmetry (2LMP, and 2M4J), i.e., 2 or 3 peptides per layer, respectively. 2M4J are AD brain-seeded fibrils while 2LMP are all-synthetic. In our long simulations of finite fibrils, all three fibril types showed fraying (unraveling) at the ends of the 6 layer fibrils; but essentially no unraveling of the infinite fibrils. In addition, the 2-fold symmetric fibril deviated less from its starting structure among the finite fibrils. All three finite structures twisted about their central axes cooperatively. In long simulations of infinite fibrils, both brain-seeded (2M4J) and the two-fold symmetrical fibrils had D23-K28 side chain salt-bridges, and these were intramolecular, intermolecular, or both (2LMP had virtually no such salt bridges). We also studied the flux of water through the fibrils. 2LMN and 2LMP structures were mostly water-tight, with the initial water molecules remaining inside internal cavities. However, pores began to form in the 2M4J infinite fibril, at the sides between peptide molecules, allowing solvent including ions to move in and out of the longitudinal core of the fibril. The pore was comprised of many of the same residues observed experimentally to have the highest rates of H/D exchange in the fibrils. This observation suggests that Ab fibrils could act as an aqueous pore that could disrupt ion fluxes if inserted into a cell membrane.
2728-Pos Board B105 Tau Mediates Widely-Spaced Microtubule Bundles through Local Polyion Attractions at the Midplane Layer: A Novel, Functional Mechanism for Intrinsically Disordered Proteins Peter J. Chung1, Chaeyeon Song1, Joanna Deek2, Herbert P. Miller3, Youli Li4, Leslie Wilson3, Stuart C. Feinstein3, Cyrus R. Safinya1. 1 Materials, Physics, and Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA, 2Cell Biophysics, Technische Universita¨t Mu¨nchen, Munich, Germany, 3 Molecular, Cell, and Developmental Biology and Neuroscience Research
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2730-Pos Board B107 Dancing with Strings: The Conformational Dynamics of VQIXXK Motifs within Tau Protein in Monomer, Fibril and Hyper-Phosphorylated Filament States Buyong Ma1, Guanghong Wei2, Jie Zhen3, Ruth Nussinov1. 1 Leidos Biomedical, NCI-Frederick, NIH, Frederick, MD, USA, 2 Department of Physics, Fudan University, Shanghai, China, 3Department of Chemical & Biomolecular Engineering, the University of Akron, Akron, OH, USA.
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2726-Pos Board B103 Self-Assembly of Full-Size Amyloid Beta 40 Proteins in Dimers Mohtadin Hashemi, Yuliang Zhang, Zhengjian Lv, Yuri L. Lyubchenko. Dept. of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE, USA. Even with growing evidence for the involvement of protein oligomeric nanoassemblies in the development of protein misfolding diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) among others, very limited knowledge exists regarding the molecular mechanisms underlying these processes. This is primarily because the protein aggregates are stabilized by weak interactions that are typically transient in nature and difficult to measure. Therefore, only mixtures of aggregates with different morphologies have been studied thus far; in turn, it is unknown how the aggregation process is initiated and how the growth of oligomers progresses. On the other hand, computational approaches can provide detailed descriptions of how monomers bind to the segments of fibrils. However, these powerful simulations, which provide the structure and dynamics of aggregates at the atomic level, require knowledge of the initial nanoassembly structure; in turn, computational analysis primarily models the elongation process of fibrils. Thus, available computational approaches can be applied to modeling the assembly of oligomers if the structure of the oligomer is known. Herein, we have conducted in silico simulations of Ab40 monomer assembly in dimers. The simulated structures were validated by comparing Monte Carlo pulling simulations with experimental data from single molecule AFM force spectroscopy enabling us to identify dimers structures rupture of which were in good agreement with the experimental results. One important observation from our long time-scale MD simulations is that the dimer does not have the extended beta-structures observed in fibrils. Rather, it remains largely unstructured and is stabilized by hydrophobic interaction and local hydrogen bonds. The results suggest that the Ab40 aggregation is a complex process in which the monomer structure depends on the aggregate size.
Institute, University of California, Santa Barbara, Santa Barbara, CA, USA, Materials Research Laboratory, University of California, Santa Barbara, Santa Barbara, CA, USA. Dysfunction of Tau, an intrinsically disordered protein localized to neuronal axons, is associated with many neurodegenerative diseases (‘‘Tauopathies’’) including FTDP-17, Alzheimer’s, and, more recently, chronic traumatic encephalopathy. However, that association is not fully understood, as the accepted, physiological functions of Tau are limited to stabilizing individual microtubules and mediating microtubule bundles. The latter conclusion is largely derived from seminal studies where transfected Tau cDNA prompted cells in culture to grow neurite-like processes with hexagonally-ordered microtubule arrays. However, ex vivo TEM of the axon shows only linear bundles of microtubules with no long-range positional ordering. Additionally, prior cell-free reconstitutions of microtubules with Tau exhibited either no bundles or bundles of dramatically different morphologies. The use of taxol in these reconstitutions further complicated matters; while making experiments tractable (stabilizing microtubules from depolymerization), taxol has been shown to weakly compete with Tau and affect microtubule assembly dynamics. Herein, we reconcile this conflicting behavior by reporting on the cell-free and taxolfree reconstitution of microtubules co-assembled with Tau at near-physiological conditions, recapitulating both hexagonally-symmetric and linear microtubule bundles. Small-angle X-ray scattering and TEM was used to obtain angstromresolution ensemble-averaged structural information and local morphology, respectively, of Tau-mediated MT bundles with large inter-MT spacings. Through an examination of truncated Tau and the force-response of microtubule bundles using osmotic depletants, we present a novel mechanism for long-ranged attractions between microtubules that is contingent on both the polyampholytic and intrinsically disordered nature of Tau. This proposed mechanism has heretofore been unseen in biology and would be significantly affected by Tau posttranslational modifications associated with dysfunction and neurodegeneration.
2727-Pos Board B104 X-Ray Digital Aggregated Dynamics of Intrinsically Disordered Proteins Naruki Hara1, Yufuku Matsushita1, Keigo Ikezaki1, Hiroshi Sekiguchi2, Naoya Fukui3, Yasushi Kawata3, Yuji C. Sasaki1,2. 1 The University of Tokyo, Chiba, Japan, 2JASRI/SPring-8, Hyogo, Japan, 3 Tottori University, Tottori, Japan. Parkinson’s Disease (PD) and Alzheimer Disease (AD) is one of the neurodegenerative pathology. These diseases are occurred by anomalous aggregations of causative proteins. Alpha-synuclein (Syn) is PD’s causative proteins and Tau is AD’s ones. But these protein’s aggregation process haven’t been clear. Additionally, these proteins are one of the famous intrinsically disorder proteins (IDP). Until now, it is difficult to understand aggregation process from static structural information of IDP. Therefore, in this study, we observed that characteristic of single molecule structural fluctuations which occurred anomalous aggregation. Furthermore, we observed dynamical digital aggregation process (from monomer to dimer or trimer). In order to understand single molecular dynamics which occurred aggregation, we measured single molecule structural fluctuations of Syn’s wild type (WT) and mutants (E46K, A53T). And, we observed digital aggregations of Tau proteins for the purpose of detecting dynamical process. At these experiments, we used Diffracted X-ray Tracking (DXT). In order to measure movements of the specific binding sites of proteins, DXT monitor X-ray diffraction spots from labeled gold nanocrystal. DXT experiments used the energy of quasi-white x-rays (energy peak-width of 2%, 10-20 keV, BL40XU, SPring-8), for that reason DXT has high time-resolution (0.1ms/frame) and high super-precision (0.1nm scale). As a result from Syn’s DXT data, The motion’s histograms in WT has simple single Gaussian distribution. However, these in other Syn’s mutants which occurred anomalous aggregation have two Gaussian distributions. From DXT results, we confirmed that mutant’s structural fluctuation is more rigid than that of WT. In the other hand, Tau dimer is very more flexible than that of monomer and trimer. The motion histograms show that there are very different process between dimerization and trimerization in the digital aggregation observations of Tau proteins.
2729-Pos Board B106 Ab Fibrils Act as Aqueous Pores: A Molecular Dynamics Study Sachin R. Natesh1, Stephen C. Meredith2, Tobin R. Sosnick3, Karl F. Freed4, Esmael J. Haddadian5. 1 Physical Sciences Collegiate Division, University of Chicago, Chicago, IL, USA, 2Dept. of Pathology and Dept. of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 3Dept. of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, USA, 4Department of Chemistry and James Frank Institute, University of Chicago, Chicago, IL, USA, 5Biological Sciences Collegiate Division, University of Chicago, Chicago, IL, USA. Alzheimer’s Disease (AD) is an increasingly prevalent neurodegenerative disease. Aggregation of Ab peptides is important in etiology of AD. We ran all-atom, explicit water molecular dynamics simulations starting from three experimentally available NMR structures of Ab fibrils, containing either 6 or an infinite number of layers. Infinite fibrils were constructed using periodic images. The NMR structures have either 2-fold (2LMN) or 3-fold rotational symmetry (2LMP, and 2M4J), i.e., 2 or 3 peptides per layer, respectively. 2M4J are AD brain-seeded fibrils while 2LMP are all-synthetic. In our long simulations of finite fibrils, all three fibril types showed fraying (unraveling) at the ends of the 6 layer fibrils; but essentially no unraveling of the infinite fibrils. In addition, the 2-fold symmetric fibril deviated less from its starting structure among the finite fibrils. All three finite structures twisted about their central axes cooperatively. In long simulations of infinite fibrils, both brain-seeded (2M4J) and the two-fold symmetrical fibrils had D23-K28 side chain salt-bridges, and these were intramolecular, intermolecular, or both (2LMP had virtually no such salt bridges). We also studied the flux of water through the fibrils. 2LMN and 2LMP structures were mostly water-tight, with the initial water molecules remaining inside internal cavities. However, pores began to form in the 2M4J infinite fibril, at the sides between peptide molecules, allowing solvent including ions to move in and out of the longitudinal core of the fibril. The pore was comprised of many of the same residues observed experimentally to have the highest rates of H/D exchange in the fibrils. This observation suggests that Ab fibrils could act as an aqueous pore that could disrupt ion fluxes if inserted into a cell membrane.
2728-Pos Board B105 Tau Mediates Widely-Spaced Microtubule Bundles through Local Polyion Attractions at the Midplane Layer: A Novel, Functional Mechanism for Intrinsically Disordered Proteins Peter J. Chung1, Chaeyeon Song1, Joanna Deek2, Herbert P. Miller3, Youli Li4, Leslie Wilson3, Stuart C. Feinstein3, Cyrus R. Safinya1. 1 Materials, Physics, and Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA, 2Cell Biophysics, Technische Universita¨t Mu¨nchen, Munich, Germany, 3 Molecular, Cell, and Developmental Biology and Neuroscience Research
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2730-Pos Board B107 Dancing with Strings: The Conformational Dynamics of VQIXXK Motifs within Tau Protein in Monomer, Fibril and Hyper-Phosphorylated Filament States Buyong Ma1, Guanghong Wei2, Jie Zhen3, Ruth Nussinov1. 1 Leidos Biomedical, NCI-Frederick, NIH, Frederick, MD, USA, 2 Department of Physics, Fudan University, Shanghai, China, 3Department of Chemical & Biomolecular Engineering, the University of Akron, Akron, OH, USA.