Determination of the Rate Constants of Oligomerization of Amyloid-β Peptides using Fluorescence...
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identified the influence from the types of FG nups and inter-molecular spacing. FG nups showed a strong dependence of inter-molecular interaction strength on molecular spacing. However, inter-molecular interaction for one type is weaker compared to that of another type. While most of the FG nups of the two types are mainly disordered despite inter- and intra- molecular interactions, we ob- served noticeable beta-sheet formation at certain inter-molecular spacings, in- dicating a significant role between inter-molecular interaction and secondary structure formation. 286-Pos Board B55 Aß Monomers Transiently Sample Oligomer and Fibril-Like Configura- tions: Ensemble Characterization using a Combined MD/NMR Approach David J. Rosenman, Christopher Connors, Wen Chen, Chunyu Wang, Angel E. Garcı ´a. Rensselaer Polytechnic Institute, Troy, NY, USA. Amyloid ß (Aß) peptides are a primary component of fibrils and oligomers im- plicated in the etiology of Alzheimer’s disease. However, the intrinsic flexibil- ity of these peptides has frustrated efforts to investigate the secondary and tertiary structure of Aß monomers, whose conformational landscapes directly contribute to the kinetics and thermodynamics of Aß aggregation. In this work, de novo replica exchange molecular dynamics (REMD) simulations on the ms/replica timescale are used to characterize the structural ensembles of Aß42, Aß40, and M35-oxidized Aß42, three physiologically prevalent isoforms with substantially different aggregation properties, the latter of which has hith- erto not been investigated with computational techniques. Further, comparisons of J coupling data calculated from the REMD trajectories with their corre- sponding experimental values are used to validate these simulations and mon- itor equilibration. Our analysis indicates that all simulations converge on the 100 ns/replica timescale toward ensembles that yield good agreement with ex- perimental J couplings. Here, we describe Aß monomers that are far more structured than other computational studies that rely on characterizations made on smaller timescales. Prominent in the C-terminus are antiparallel ß-hairpin topologies between L17-A21, A30-L36, and V39-A41, reminiscent of oligomer and fibril models, that expose the composite hydrophobic side chains to solvent and may serve as hotspots for self-association. A persistent V24-K28 bend motif is observed in all three species that is stabilized by buried backbone to side chain hydrogen bonds with D23 and a cross-region salt bridge between E22 and K28, highlighting the role of the side chain identities of the FAD-linked E22 and D23 residues in Aß monomer structure. These character- izations help illustrate the conformational landscapes of Aß monomers at atomic resolution and provide insight into the early stages of Aß aggregation pathways. 287-Pos Board B56 The Functional Dependence of the Intrinsically Disordered Domains of Tau Peter J. Chung 1 , Joanna Deek 1 , H. Eric Feinstein 1 , M.C. Choi 2 , Leslie Wilson 1 , Stuart Feinstein 1 , Cyrus R. Safinya 1 . 1 University of California, Santa Barbara, Santa Barbara, CA, USA, 2 Korean Advanced Institute of Science and Technology, Deajon, Korea, Republic of. Hyperphosphorylated tau is the precursor to neurofibrillary tangles, one of the hallmark symptoms of Alzheimer’s disease [1]. In its non-diseased state, the microtubule-associated protein tau promotes the assembly of tubulin into mi- crotubules and regulates axonal transport. However, dysfunction of tau has been implicated in a class of diseases known as tauopathies, of which Alz- heimer’s is the most pervasive [2]. Characterization of tau has proven to be dif- ficult, due to the presence of intrinsically disordered domains in the N- and C-termini of tau which precludes protein crystallography [3]. Using solution small-angle x-ray scattering, we examined tau-induced bundles of microtubules in cell-free conditions. By this method, we directly measured the inter- microtubule spacing by tau, the magnitude of the attractive force of tau, and the domains of the N- and C- termini that are responsible for the bundling of microtubules. The six isoforms of tau are the result of alternative splicing and are commonly described as sequentially having an N-terminal (consisting of a projection do- main and proline-rich region), microtubule-binding repeats, and a C-terminal. To examine the functional dependence of the projection domain, proline- rich region, and C-terminal, distinct tau constructs were expressed with deleted domains. Using the wild-type isoforms, the inter-microtubule spacing was found to be modulated by the length of the projection domain. Surpris- ingly, the use of the deletion constructs showed that neither the projection- domain or proline-rich region is necessary for bundling, in conflict with existing models [4]. Funded by DOE-BES-DOE-DE-FG02-06ER46314 and NSF-DMR-1101900 1. J.G. Wood et. al. PNAS. 83; 4040 (1986). 2. B. Esmaeli-Azad, J.H. McCarty, and S.C. Feinstein. J. Cell Sci. 107; 869 (1994). 3. H. Wille, G. Drewes, J. Biernat, E.-M. Mandelkow, and E. Mandelkow. JCB. 188; 573 (1992). 4. Y. Kanai et. al. JCB. 109; 1173 (1989). 288-Pos Board B57 Determination of the Rate Constants of Oligomerization of Amyloid-b Peptides using Fluorescence Quenching of Tetramethylrhodamine Kanchan Garai, Carl Frieden. Washington University School of Medicine, St. Louis, MO, USA. Soluble oligomers of amyloid-b (Ab) play a central role in the pathology of Alzheimer’s disease. Here we have determined the rate constants of the monomer-oligomer process of Ab using a new method based on self- quenching of tetramethylrhodamine (TMR) which is covalently labeled at the N-terminal of Ab. During aggregation TMR fluorescence is quenched in a time dependent manner in three distinct phases, an early oligomerization phase, a lag phase and a growth phase. Kinetic data of the early phase are consistent with a monomer-dimer-trimer process. The rate and the equilibrium constants differ markedly between Ab 1-42 and Ab 1-40 showing higher oligomerization propen- sity for A 1-42 .The association rate constants (~10 2 M 1 s 1 ) for both the peptides are slow compared to a diffusion limited process. The rates of oligomerization are al- tered dramatically with increase of temper- ature and are highly sensitive to changes in pH especially below pH 7.5. Our results for the first time provide quantitative esti- mation of the kinetics of monomer- oligomer process of Ab. The methodologies presented here are simple and therefore can be easily applied to other amyloidogenic proteins. 289-Pos Board B58 Inhibition and Mechanism of Islet Amyloid Polypeptide Toxicity Diana E. Schlamadinger, Sunil Kumar, Mazin Magzoub, James A. Hebda, Andrew D. Miranker. Yale University, New Haven, CT, USA. A critical aspect of type II diabetes is the dysfunction and death of insulin- secreting b-cells. A 37-residue peptide hormone, islet amyloid polypeptide (IAPP), plays a central role in this pathology since it is known that rodent models transgenic for human IAPP show spontaneous development of b-cell dysfunction and diabetic symptoms. Recent evidence suggests that a subset of several alternative a-helical membrane-bound oligomers on-pathway to am- yloid fiber formation, and not the amyloid fibrils themselves, are the toxic spe- cies in disease. We recently showed that IAPP adopts cell and membrane penetrating properties that are likely correlated with its toxicity towards b-cells; IAPP localizes to lysosomes of rat insulinoma pancreas b-cells (INS-1) at pep- tide concentrations determined by cell viability assays to be nontoxic, and re- locates to mitochondria at toxic peptide concentrations (M. Magzoub and A. D. Miranker, Faseb J., 2011, 26, 1228-1238). Here, we evaluate the ability of small molecules to agonize and antagonize IAPP toxicity towards INS-1 cells via large-scale screens of cell viability. Localization of IAPP to subcellular compartments of INS-1 cells in the presence of these molecules is also inves- tigated. We further describe the activity modulation and subcellular localiza- tion of the peptide in the presence of small molecules from libraries at Yale Small Molecule Discovery Center that have been shown to slow lipid- catalyzed amyloid fiber formation of IAPP. 290-Pos Board B59 Screening and Design of New Modulators of IAPP Membrane-Binding and Toxicity Abhinav Nath, Diana E. Schlamadinger, Shenil Dodhia, Elizabeth Rhoades, Andrew D. Miranker. Yale University, New Haven, CT, USA. Type 2 Diabetes Mellitus (T2DM) is characterized by pancreatic amyloid ag- gregates composed of islet amyloid polypeptide (IAPP; also called amylin), a small, intrinsically disordered peptide hormone. Pancreatic b-cell death due to membrane disruption mediated by non-amyloid or pre-amyloid oligomers of IAPP is thought to be central to the progression of T2DM. However, the ra- tional design and screening of small-molecule and peptide ligands targeting these oligomeric states of IAPP is challenging using current pharmacological and biophysical methods. We recently used intermolecular single-pair Fo ¨rster resonance energy transfer (spFRET) combined with constrained Monte Carlo 56a Sunday, February 3, 2013
Transcript of Determination of the Rate Constants of Oligomerization of Amyloid-β Peptides using Fluorescence...
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identified the influence from the types of FG nups and inter-molecular spacing.FG nups showed a strong dependence of inter-molecular interaction strength onmolecular spacing. However, inter-molecular interaction for one type is weakercompared to that of another type. While most of the FG nups of the two typesare mainly disordered despite inter- and intra- molecular interactions, we ob-served noticeable beta-sheet formation at certain inter-molecular spacings, in-dicating a significant role between inter-molecular interaction and secondarystructure formation.
286-Pos Board B55Aß Monomers Transiently Sample Oligomer and Fibril-Like Configura-tions: Ensemble Characterization using a Combined MD/NMR ApproachDavid J. Rosenman, Christopher Connors, Wen Chen, Chunyu Wang,Angel E. Garcıa.Rensselaer Polytechnic Institute, Troy, NY, USA.Amyloid ß (Aß) peptides are a primary component of fibrils and oligomers im-plicated in the etiology of Alzheimer’s disease. However, the intrinsic flexibil-ity of these peptides has frustrated efforts to investigate the secondary andtertiary structure of Aß monomers, whose conformational landscapes directlycontribute to the kinetics and thermodynamics of Aß aggregation. In thiswork, de novo replica exchange molecular dynamics (REMD) simulations onthe ms/replica timescale are used to characterize the structural ensembles ofAß42, Aß40, andM35-oxidized Aß42, three physiologically prevalent isoformswith substantially different aggregation properties, the latter of which has hith-erto not been investigated with computational techniques. Further, comparisonsof J coupling data calculated from the REMD trajectories with their corre-sponding experimental values are used to validate these simulations and mon-itor equilibration. Our analysis indicates that all simulations converge on the100 ns/replica timescale toward ensembles that yield good agreement with ex-perimental J couplings. Here, we describe Aß monomers that are far morestructured than other computational studies that rely on characterizationsmade on smaller timescales. Prominent in the C-terminus are antiparallelß-hairpin topologies between L17-A21, A30-L36, and V39-A41, reminiscentof oligomer and fibril models, that expose the composite hydrophobic sidechains to solvent and may serve as hotspots for self-association. A persistentV24-K28 bend motif is observed in all three species that is stabilized by buriedbackbone to side chain hydrogen bonds with D23 and a cross-region salt bridgebetween E22 and K28, highlighting the role of the side chain identities of theFAD-linked E22 and D23 residues in Aß monomer structure. These character-izations help illustrate the conformational landscapes of Aß monomers atatomic resolution and provide insight into the early stages of Aß aggregationpathways.
287-Pos Board B56The Functional Dependence of the Intrinsically Disordered Domainsof TauPeter J. Chung1, Joanna Deek1, H. Eric Feinstein1, M.C. Choi2,Leslie Wilson1, Stuart Feinstein1, Cyrus R. Safinya1.1University of California, Santa Barbara, Santa Barbara, CA, USA, 2KoreanAdvanced Institute of Science and Technology, Deajon, Korea, Republic of.Hyperphosphorylated tau is the precursor to neurofibrillary tangles, one of thehallmark symptoms of Alzheimer’s disease [1]. In its non-diseased state, themicrotubule-associated protein tau promotes the assembly of tubulin into mi-crotubules and regulates axonal transport. However, dysfunction of tau hasbeen implicated in a class of diseases known as tauopathies, of which Alz-heimer’s is the most pervasive [2]. Characterization of tau has proven to be dif-ficult, due to the presence of intrinsically disordered domains in the N- andC-termini of tau which precludes protein crystallography [3]. Using solutionsmall-angle x-ray scattering, we examined tau-induced bundles of microtubulesin cell-free conditions. By this method, we directly measured the inter-microtubule spacing by tau, the magnitude of the attractive force of tau, andthe domains of the N- and C- termini that are responsible for the bundling ofmicrotubules.The six isoforms of tau are the result of alternative splicing and are commonlydescribed as sequentially having an N-terminal (consisting of a projection do-main and proline-rich region), microtubule-binding repeats, and a C-terminal.To examine the functional dependence of the projection domain, proline-rich region, and C-terminal, distinct tau constructs were expressed with deleteddomains. Using the wild-type isoforms, the inter-microtubule spacingwas found to be modulated by the length of the projection domain. Surpris-ingly, the use of the deletion constructs showed that neither the projection-domain or proline-rich region is necessary for bundling, in conflict withexisting models [4].Funded by DOE-BES-DOE-DE-FG02-06ER46314 and NSF-DMR-11019001. J.G. Wood et. al. PNAS. 83; 4040 (1986).
2. B. Esmaeli-Azad, J.H. McCarty, and S.C. Feinstein. J. Cell Sci. 107; 869(1994).3. H. Wille, G. Drewes, J. Biernat, E.-M. Mandelkow, and E. Mandelkow. JCB.188; 573 (1992).4. Y. Kanai et. al. JCB. 109; 1173 (1989).
288-Pos Board B57Determination of the Rate Constants of Oligomerization of Amyloid-bPeptides using Fluorescence Quenching of TetramethylrhodamineKanchan Garai, Carl Frieden.Washington University School of Medicine, St. Louis, MO, USA.Soluble oligomers of amyloid-b (Ab) play a central role in the pathology ofAlzheimer’s disease. Here we have determined the rate constants of themonomer-oligomer process of Ab using a new method based on self-quenching of tetramethylrhodamine (TMR) which is covalently labeled atthe N-terminal of Ab. During aggregation TMR fluorescence is quenched in atime dependent manner in three distinct phases, an early oligomerization phase,a lag phase and a growth phase.Kinetic data of the early phase are consistentwitha monomer-dimer-trimer process. The rate and the equilibrium constants differmarkedly between Ab1-42 and Ab1-40 showing higher oligomerization propen-sity for A1-42.The association rate constants (~10
2M�1s�1) for both the peptides
are slow compared to a diffusion limitedprocess. The rates of oligomerization are al-tered dramatically with increase of temper-ature and are highly sensitive to changes inpH especially below pH 7.5. Our resultsfor the first time provide quantitative esti-mation of the kinetics of monomer-oligomer process of Ab. Themethodologiespresented here are simple and therefore canbe easily applied to other amyloidogenicproteins.289-Pos Board B58Inhibition and Mechanism of Islet Amyloid Polypeptide ToxicityDiana E. Schlamadinger, Sunil Kumar, Mazin Magzoub, James A. Hebda,Andrew D. Miranker.Yale University, New Haven, CT, USA.A critical aspect of type II diabetes is the dysfunction and death of insulin-secreting b-cells. A 37-residue peptide hormone, islet amyloid polypeptide(IAPP), plays a central role in this pathology since it is known that rodentmodels transgenic for human IAPP show spontaneous development of b-celldysfunction and diabetic symptoms. Recent evidence suggests that a subsetof several alternative a-helical membrane-bound oligomers on-pathway to am-yloid fiber formation, and not the amyloid fibrils themselves, are the toxic spe-cies in disease. We recently showed that IAPP adopts cell and membranepenetrating properties that are likely correlated with its toxicity towards b-cells;IAPP localizes to lysosomes of rat insulinoma pancreas b-cells (INS-1) at pep-tide concentrations determined by cell viability assays to be nontoxic, and re-locates to mitochondria at toxic peptide concentrations (M. Magzoub and A. D.Miranker, Faseb J., 2011, 26, 1228-1238). Here, we evaluate the ability ofsmall molecules to agonize and antagonize IAPP toxicity towards INS-1 cellsvia large-scale screens of cell viability. Localization of IAPP to subcellularcompartments of INS-1 cells in the presence of these molecules is also inves-tigated. We further describe the activity modulation and subcellular localiza-tion of the peptide in the presence of small molecules from libraries at YaleSmall Molecule Discovery Center that have been shown to slow lipid-catalyzed amyloid fiber formation of IAPP.
290-Pos Board B59Screening and Design of NewModulators of IAPPMembrane-Binding andToxicityAbhinav Nath, Diana E. Schlamadinger, Shenil Dodhia, Elizabeth Rhoades,Andrew D. Miranker.Yale University, New Haven, CT, USA.Type 2 Diabetes Mellitus (T2DM) is characterized by pancreatic amyloid ag-gregates composed of islet amyloid polypeptide (IAPP; also called amylin),a small, intrinsically disordered peptide hormone. Pancreatic b-cell death dueto membrane disruption mediated by non-amyloid or pre-amyloid oligomersof IAPP is thought to be central to the progression of T2DM. However, the ra-tional design and screening of small-molecule and peptide ligands targetingthese oligomeric states of IAPP is challenging using current pharmacologicaland biophysical methods. We recently used intermolecular single-pair Forsterresonance energy transfer (spFRET) combined with constrained Monte Carlo
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