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Tau Binds to Multiple Tubulin Dimers with Helical Structure
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Wednesday, March 2, 2016
Intrinsically disordered protein (IDP) of tau binds and stabilizes microtubule, which contributes to the proper function of neuron, while its aggregation is implicated in Alzheimer’s disease. In recent years, we have conducted various extensive molecular dynamics simulations of tau proteins in their monomer, normal fibril, and hyper-phosphorylated filament states. The conformations of two critical hexapeptides (275VQIINK280 and 306VQIVYK311) have changed from random, alpha-helical, and beta-sheets, in concert with overall conformational changes of tau protein. In the isolated monomeric states, we observed the dynamically ordered structures are evolved from disordered conformations. Our REMD simulations revealed the structural diversity of K18 and K19 monomers, including helix-rich and mixed helix- and beta-sheetrich structures. The two VQIXXK motifs have high beta-sheet contents and large hydrophobic surface exposure. The preformed Ab1-42 protofibril can stretch tau conformation, and drastically reduces the metastable secondary structures/hydrogen bonding/salt-bridge networks in tau monomers, and exposes VQIXXK motifs more. In tau amyloid fibril, the VQIXXK motifs can be embedded tightly in linear or bent beta-sheet motifs. However, when N- and C-terminals of tau protein are highly phosphorylated, the VQIXXK motifs in amyloid fibril may have more solvent exposure. References: (1) J. Phys. Chem. Lett., 2015, 6 (16), pp 3276-3282. (2) J Phys Chem Lett. 2014, 4;5(17):3026-3031. (3) Chem Commun. 2013 4;49(34):3582-4. (4) J Am Chem Soc. 2012, 20;134(24):10271-8. (5) J Biol Chem. 2012, 27;287(18):14950-9. 2731-Pos Board B108 The Conformation of Ab-Peptide Aggregates on 2D Surfaces is Different than in Solution: A Molecular Dynamics Study Sachin R. Natesh1, Kark F. Freed2, Esmael J. Haddadian3. 1 Physical Sciences Collegiate Divisin, University of Chicago, Chicago, IL, USA, 2Department of Chemistry and James Frank Institute, University of Chicago, Chicago, IL, USA, 3Biological Sciences Collegiate Divisin, University of Chicago, Chicago, IL, USA. Alzheimer’s Disease (AD) is an important and increasingly prevalent neurodegenerative disease. Aggregation of Ab-peptides is important in etiology of AD. It has been shown experimentally that Ab-peptides self assemble to form fibrils on 2D surfaces at much faster rates than they form fibrils in the 3D environment of solutions. However, the molecular mechanism of the fibril formation and the conformation of the fibril on 2D surfaces remain unknown. We have run long molecular dynamics simulations of Ab (1-40) monomers on Alkanethiol selfassembled monolayers (SAM) with different functional head groups and also in bulk water solution. We began with Ab-peptides pre-adsorbed on ˚ from the surface). SAM-CH3 and -COOH restricted Ab-peptide SAMs (~ 5A motion, leading to inter-monomer b-strand formation within orders of 100s of nanoseconds. The same effect was not observed in bulk solution. Unlike the SAM-CH3 and -COOH surfaces, Ab-monomer adsorption on the SAM-OH surface was weak, causing monomers to leave the surface and move into solution. These results indicate the importance of the combination of hydrophobic and electrostatic interactions for mediating Ab self-assembly on the SAM. A simu˚ above the SAM-CH3 surface lation in which Ab-peptides were initiated 15A further supported our result. Ab-peptides initially showed low b-content, but once they were adsorbed (~ 170 ns) the b-content increased to values similar to our simulations of pre-adsorbed monomers. The b-sheet formation was both inter- and intra-peptide though mostly parallel to the surface in contrast to the NMR data in bulk solution where the Ab-monomers stacked on top of each other. The residues involved in b-sheet formation were somewhat different from those observed in the NMR data. These observations suggested that Ab-peptides could possibly blanket the cell surface disrupting cell communication with the outside. 2732-Pos Board B109 Tau Binds to Multiple Tubulin Dimers with Helical Structure Xiaohan Li1, Jacob A. Culver2, Elizabeth Rhoades3. 1 Chemistry, Yale University, New Haven, CT, USA, 2Ball State University, Muncie, IN, USA, 3Chemistry, University of Pennsylvania, Philadelphia, CT, USA. Tau is a microtubule associated protein (MAP) which functions to maintain stability of microtubule as well as promote microtubule (MT) assembly in the axons of neurons. Understanding the mechanism of tau-promoted microtubule polymerization may provide insight into its loss of function contributing to its pathology in neurodegenerative diseases. The interaction between tau and tubulin dimers plays an important part in tau-promoted microtubule polymerization. However, study of this interaction has been hindered by tau’s intrinsically disordered nature and the highly dynamic nature of tubulin and MTs. Here, with a combination of fluorescence correlation spectroscopy (FCS) and acrylodan fluorescence screening, we show that tau binds to multiple tubulin
dimers, even in the presence of molecules which block MT assembly. Moreover, we observe a disorder-to-helical transition in the microtubule binding region of tau upon binding. Our findings support a model where the intrinsic disorder and disorder to order transition of tau provides a flexible scaffold for mediating functional interactions between tau and tubulin or MTs. 2733-Pos Board B110 The Disparate Effects of two Molecular Chaperones on Tau Amyloid Formation Hannah E.R. Baughman1, Amanda F. Clouser2, Rachel E. Klevit2, Abhinav Nath1. 1 Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA, 2Department of Biochemistry, University of Washington, Seattle, WA, USA. Tau is an intrinsically disordered protein that binds microtubules and plays important roles in axonal transport and microtubule stability in neurons. It has been implicated in a set of neurodegenerative diseases collectively termed tauopathies, characterized by the dissociation of tau from microtubules and its accumulation in pathological, insoluble amyloid-type aggregates. Molecular chaperones are important players in all amyloid diseases, including tauopathies, as they are part of the cellular response to prevent protein misfolding and aggregation. Tau has been shown to interact with numerous chaperones, although much of the previous work is biological in nature and few interactions have been characterized quantitatively at the molecular level. We seek to better define the interactions between tau and two heat shock proteins, Hsc70 and HSPB1. While both proteins act to prevent tau aggregation and likely play protective roles in tauopathies, their mechanisms of action are distinct. Hsc70 is a 71 kDa ATPase that uses the energy of ATP hydrolysis to assist client proteins to fold to their native structures. In contrast, HSPB1 is a 27 kDa small heat shock protein that forms large, heterogeneous oligomers and functions as a holdase, binding improperly-folded proteins and preventing aggregation. Using NMR spectroscopy, electron microscopy, fluorescence correlation spectroscopy, and other biophysical tools, we characterized the modes by which each protein binds tau and the effects of each protein on the kinetics of tau fibril formation. We show that each protein binds the aggregation-prone microtubule binding repeat region of tau, but they recognize distinct sequences within this region. In addition, the two chaperone proteins have inhibitory effects on fibril formation, but HSPB1 acts primarily during the nucleation phase of fibril formation, whereas Hsc70 has a greater effect on fibril elongation. 2734-Pos Board B111 Binding-Activated Superradiant Probes for Amyloid in Solution and Tissue Patrick Donabedian1,2, Nicole Maphis3, Shanya Jiang3, Kiran Bhaskar3, David Whitten2,4, Eva Chi2,4. 1 Nanoscience and Microsystems Engineering, University of New Mexico, Albuquerque, NM, USA, 2Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM, USA, 3Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM, USA, 4 Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM, USA. Improved fluorescent detection of amyloid protein aggregates would accelerate research into the biology of amyloid formation, implicated in Alzheimer’s and Parkinson’s diseases, among other neurodegenerative conditions. We report the development of a fluorescent sensor OPE1 for amyloids, competent for oneand two-photon imaging, with a strong binding-activated increase in emission based on formation of superluminescent J aggregates and dequenching in a hydrophobic environment. OPE1 stains neurofibrillary tangles and beta-amyloid plaques with low background in murine and human brain tissue, and detects a wide variety of in vitro-formed amyloid aggregates. Unlike conventional sensors such as thioflavin T, OPE1 exhibits static quenching in water based on interactions with chromophore-attached ethyl ester moieties as well as a propensity to form highly emissive J-type aggregates. We provide evidence that both these mechanisms are at work in the large increase in fluorescence intensity observed for OPE1 with amyloid fibrils. We hope that these novel mechanisms will lead to the development of improved self-assembling fluorescent probes for amyloid and other relevant analytes. 2735-Pos Board B112 Amyloid Aggregation of Amylin: Gain of Function along Aggregation Pathway? Anoop Rawat, Debanjan Bhowmik, Barun Kumar Maity, Sudipta Maiti. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. Abstract: Aggregation of human amylin, a 37 amino acid residue neuropeptide of pancreatic origin, into amyloid aggregates is implicated in the etiology of diabetes mellitus type II. Despite its clinical significance, details of progression
Wednesday, March 2, 2016
Intrinsically disordered protein (IDP) of tau binds and stabilizes microtubule, which contributes to the proper function of neuron, while its aggregation is implicated in Alzheimer’s disease. In recent years, we have conducted various extensive molecular dynamics simulations of tau proteins in their monomer, normal fibril, and hyper-phosphorylated filament states. The conformations of two critical hexapeptides (275VQIINK280 and 306VQIVYK311) have changed from random, alpha-helical, and beta-sheets, in concert with overall conformational changes of tau protein. In the isolated monomeric states, we observed the dynamically ordered structures are evolved from disordered conformations. Our REMD simulations revealed the structural diversity of K18 and K19 monomers, including helix-rich and mixed helix- and beta-sheetrich structures. The two VQIXXK motifs have high beta-sheet contents and large hydrophobic surface exposure. The preformed Ab1-42 protofibril can stretch tau conformation, and drastically reduces the metastable secondary structures/hydrogen bonding/salt-bridge networks in tau monomers, and exposes VQIXXK motifs more. In tau amyloid fibril, the VQIXXK motifs can be embedded tightly in linear or bent beta-sheet motifs. However, when N- and C-terminals of tau protein are highly phosphorylated, the VQIXXK motifs in amyloid fibril may have more solvent exposure. References: (1) J. Phys. Chem. Lett., 2015, 6 (16), pp 3276-3282. (2) J Phys Chem Lett. 2014, 4;5(17):3026-3031. (3) Chem Commun. 2013 4;49(34):3582-4. (4) J Am Chem Soc. 2012, 20;134(24):10271-8. (5) J Biol Chem. 2012, 27;287(18):14950-9. 2731-Pos Board B108 The Conformation of Ab-Peptide Aggregates on 2D Surfaces is Different than in Solution: A Molecular Dynamics Study Sachin R. Natesh1, Kark F. Freed2, Esmael J. Haddadian3. 1 Physical Sciences Collegiate Divisin, University of Chicago, Chicago, IL, USA, 2Department of Chemistry and James Frank Institute, University of Chicago, Chicago, IL, USA, 3Biological Sciences Collegiate Divisin, University of Chicago, Chicago, IL, USA. Alzheimer’s Disease (AD) is an important and increasingly prevalent neurodegenerative disease. Aggregation of Ab-peptides is important in etiology of AD. It has been shown experimentally that Ab-peptides self assemble to form fibrils on 2D surfaces at much faster rates than they form fibrils in the 3D environment of solutions. However, the molecular mechanism of the fibril formation and the conformation of the fibril on 2D surfaces remain unknown. We have run long molecular dynamics simulations of Ab (1-40) monomers on Alkanethiol selfassembled monolayers (SAM) with different functional head groups and also in bulk water solution. We began with Ab-peptides pre-adsorbed on ˚ from the surface). SAM-CH3 and -COOH restricted Ab-peptide SAMs (~ 5A motion, leading to inter-monomer b-strand formation within orders of 100s of nanoseconds. The same effect was not observed in bulk solution. Unlike the SAM-CH3 and -COOH surfaces, Ab-monomer adsorption on the SAM-OH surface was weak, causing monomers to leave the surface and move into solution. These results indicate the importance of the combination of hydrophobic and electrostatic interactions for mediating Ab self-assembly on the SAM. A simu˚ above the SAM-CH3 surface lation in which Ab-peptides were initiated 15A further supported our result. Ab-peptides initially showed low b-content, but once they were adsorbed (~ 170 ns) the b-content increased to values similar to our simulations of pre-adsorbed monomers. The b-sheet formation was both inter- and intra-peptide though mostly parallel to the surface in contrast to the NMR data in bulk solution where the Ab-monomers stacked on top of each other. The residues involved in b-sheet formation were somewhat different from those observed in the NMR data. These observations suggested that Ab-peptides could possibly blanket the cell surface disrupting cell communication with the outside. 2732-Pos Board B109 Tau Binds to Multiple Tubulin Dimers with Helical Structure Xiaohan Li1, Jacob A. Culver2, Elizabeth Rhoades3. 1 Chemistry, Yale University, New Haven, CT, USA, 2Ball State University, Muncie, IN, USA, 3Chemistry, University of Pennsylvania, Philadelphia, CT, USA. Tau is a microtubule associated protein (MAP) which functions to maintain stability of microtubule as well as promote microtubule (MT) assembly in the axons of neurons. Understanding the mechanism of tau-promoted microtubule polymerization may provide insight into its loss of function contributing to its pathology in neurodegenerative diseases. The interaction between tau and tubulin dimers plays an important part in tau-promoted microtubule polymerization. However, study of this interaction has been hindered by tau’s intrinsically disordered nature and the highly dynamic nature of tubulin and MTs. Here, with a combination of fluorescence correlation spectroscopy (FCS) and acrylodan fluorescence screening, we show that tau binds to multiple tubulin
dimers, even in the presence of molecules which block MT assembly. Moreover, we observe a disorder-to-helical transition in the microtubule binding region of tau upon binding. Our findings support a model where the intrinsic disorder and disorder to order transition of tau provides a flexible scaffold for mediating functional interactions between tau and tubulin or MTs. 2733-Pos Board B110 The Disparate Effects of two Molecular Chaperones on Tau Amyloid Formation Hannah E.R. Baughman1, Amanda F. Clouser2, Rachel E. Klevit2, Abhinav Nath1. 1 Department of Medicinal Chemistry, University of Washington, Seattle, WA, USA, 2Department of Biochemistry, University of Washington, Seattle, WA, USA. Tau is an intrinsically disordered protein that binds microtubules and plays important roles in axonal transport and microtubule stability in neurons. It has been implicated in a set of neurodegenerative diseases collectively termed tauopathies, characterized by the dissociation of tau from microtubules and its accumulation in pathological, insoluble amyloid-type aggregates. Molecular chaperones are important players in all amyloid diseases, including tauopathies, as they are part of the cellular response to prevent protein misfolding and aggregation. Tau has been shown to interact with numerous chaperones, although much of the previous work is biological in nature and few interactions have been characterized quantitatively at the molecular level. We seek to better define the interactions between tau and two heat shock proteins, Hsc70 and HSPB1. While both proteins act to prevent tau aggregation and likely play protective roles in tauopathies, their mechanisms of action are distinct. Hsc70 is a 71 kDa ATPase that uses the energy of ATP hydrolysis to assist client proteins to fold to their native structures. In contrast, HSPB1 is a 27 kDa small heat shock protein that forms large, heterogeneous oligomers and functions as a holdase, binding improperly-folded proteins and preventing aggregation. Using NMR spectroscopy, electron microscopy, fluorescence correlation spectroscopy, and other biophysical tools, we characterized the modes by which each protein binds tau and the effects of each protein on the kinetics of tau fibril formation. We show that each protein binds the aggregation-prone microtubule binding repeat region of tau, but they recognize distinct sequences within this region. In addition, the two chaperone proteins have inhibitory effects on fibril formation, but HSPB1 acts primarily during the nucleation phase of fibril formation, whereas Hsc70 has a greater effect on fibril elongation. 2734-Pos Board B111 Binding-Activated Superradiant Probes for Amyloid in Solution and Tissue Patrick Donabedian1,2, Nicole Maphis3, Shanya Jiang3, Kiran Bhaskar3, David Whitten2,4, Eva Chi2,4. 1 Nanoscience and Microsystems Engineering, University of New Mexico, Albuquerque, NM, USA, 2Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM, USA, 3Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM, USA, 4 Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM, USA. Improved fluorescent detection of amyloid protein aggregates would accelerate research into the biology of amyloid formation, implicated in Alzheimer’s and Parkinson’s diseases, among other neurodegenerative conditions. We report the development of a fluorescent sensor OPE1 for amyloids, competent for oneand two-photon imaging, with a strong binding-activated increase in emission based on formation of superluminescent J aggregates and dequenching in a hydrophobic environment. OPE1 stains neurofibrillary tangles and beta-amyloid plaques with low background in murine and human brain tissue, and detects a wide variety of in vitro-formed amyloid aggregates. Unlike conventional sensors such as thioflavin T, OPE1 exhibits static quenching in water based on interactions with chromophore-attached ethyl ester moieties as well as a propensity to form highly emissive J-type aggregates. We provide evidence that both these mechanisms are at work in the large increase in fluorescence intensity observed for OPE1 with amyloid fibrils. We hope that these novel mechanisms will lead to the development of improved self-assembling fluorescent probes for amyloid and other relevant analytes. 2735-Pos Board B112 Amyloid Aggregation of Amylin: Gain of Function along Aggregation Pathway? Anoop Rawat, Debanjan Bhowmik, Barun Kumar Maity, Sudipta Maiti. Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India. Abstract: Aggregation of human amylin, a 37 amino acid residue neuropeptide of pancreatic origin, into amyloid aggregates is implicated in the etiology of diabetes mellitus type II. Despite its clinical significance, details of progression