Characterizing a -Synuclein Membrane Bound Structure

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Characterizing a -Synuclein Membrane Bound Structure. Bert Lai (Gray Group) Department of Chemistry and Chemical Engineering California Institute of Technology Thesis Defense 04/29/08. Introduction: α-Synuclein. Role of α -syn in Parkinson’s disease pathogenesis unclear - PowerPoint PPT Presentation

Transcript of Characterizing a -Synuclein Membrane Bound Structure

  • Characterizing a-Synuclein Membrane Bound StructureBert Lai (Gray Group)Department of Chemistry and Chemical EngineeringCalifornia Institute of Technology

    Thesis Defense04/29/08

  • Introduction: -Synuclein Role of -syn in Parkinsons disease pathogenesis unclear Aggregates form amyloid fibrils major component of Lewy Bodies deposits found in substantia nigra of Parkinsons disease victims Three single point mutations (A30P, A53T, and E46K) Early onset familial Parkinsons diseaseSubstantia nigraParkinsons DiseaseNormal

  • -Synuclein SequenceThree interesting regions:Non -Amyloid Component (NAC) region (Residues 61-95)Commonly found in Alzheimers disease aggregates Acidic C-terminal region (Residues 96-140)7 imperfect repeats of 11 amino acids Second and seventh residues of each repeat are mostly KTKEGV Typically found in apolipoproteins

    Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595-9603.

  • Synaptic vesicle-associated protein Oligomers of -syn involved in membrane permeabilization Structure: In solution: unfolded with structural preference In SDS micelles/acidic small unilamellar vesicles (SUVs): highly helical

    Introduction: -Synuclein and Membrane SDS micellesSUVs1 nm20-50 nm

  • Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595-9603.NMR Model of -Synuclein in Micelles Formation of -helices at residue 3 to 37 (1st helix) residue 45 to 92 (2nd helix) Formation of linker between 2 helices Unstructured hydrophilic tail

  • Phospholipid VesiclesEPR: Helical structure in the 7 N-terminal 11-amino acid repeatsJao, C. C.; Der-Sarkissian, A.; Chen, J.; Langen, R. Proc Natl. Acad. Sci. 2004, 101 (22), 8331-8336.Lipid exposedSolvent exposed

  • The CD spectra show that acidic lipid is needed for -syn to form helical structures.Phospholipids1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC)1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphate (POPA)1:1 POPC:POPA SUVs were made

  • For the N-terminus

  • Trp-Tyr(NO2) pairs494ro = 26 10 < r < 40 W4-Y94Helix #1Helix #2494

  • Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124 (19), 5481-5485.FET Kinetics & Polypeptide Conformations

  • Steady State Fluorescence StudiesIntensityW4 is in a more hydrophobic environment when associated with SDS or SUVWavelength (nm)Amount of quenching by Y(NO2): SDS > SUV > Buffer

  • 4The two helices are closer together in SDS micelles.NaPiSDSSUVDistance ()P(r)80 %90 %40 %Time-resolved Fluorescence StudiesTime (ns)It

  • Summary Extended helix is elucidated when -syn is in association with SUVs Similar to the EPR model -syn forms compact structures when SDS micelles are introduced Similar to the NMR modelSome extended structure remains

  • Calcium Binding Behavior of a-Synucleins C-Terminal Tail

  • C-terminal Tail Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Mol. Biol. 2005, 280, 9595-9603. locates in residues 96-140 is unstructured in the presence of membrane mimic is highly negatively charged (10 Glus & 5 Asps) modulates aggregation binds to metal ions, e.g. calcium, copper microtubule-associated proteins 1B polyamines

    Aging Oxidative Stress Calcium Dysregulation

  • Tamamizu-Kato, S.; Kosaraju, M. G.; Kato, H.; Raussens, V.; Ruysschaert, J.-M.; Narayanaswami, V. Biochemistry 2006, 45, 10947-.de Laureto, P. P.; Tosatto, L.; Frane, E.; Marin, O.; Uversky, V. N.; Fontana, A. Biochemistry 2006, 45, 38, 11523-.Theory 1: C-terminal tail becomes -sheet Theory 2: C-terminal tail rigidified by binding calcium ion

    Study C-terminal tail behavior in the presence of POPC:POPA with various Ca2+ concentrationStage 1: Trp-only mutantsStage 2: Donor-acceptor pair

  • DNP (N-Dinitrophenyl Phosphatidylethanolamine)

    Br (6,7) POPC (1-Palmitoyl-2-Stearoyl(6,7-dibromo)-sn-Glycero-3-Phosphocholine)

    McIntosh, T. J.; Holloway, P. W. Biochemistry 1987, 26, 1783-1788.Quencher located on the end group94 101 125 136 Headgroup/hydrocarbon boundary and quencher:3.5

  • Trp Only Steady-state FluorescenceNATAW125W101W136Calcium Titration Curves (in SUVs)[Ca2+][Ca2+][Ca2+][Ca2+]

  • CD shows calcium ion does not affect the structure of -synuclein.CDTime-resolved Fluorescent LifetimesW101 seems to be more associated with the membrane, but not deeply inserted.

  • W101 DNP Fluorescence Transfer Rate DNP + Ca2+DNPA shift to shorter lifetimes is observed when calcium ions were added.P(r)Log(k)

  • Tamamizu-Kato, S.; Kosaraju, M. G.; Kato, H.; Raussens, V.; Ruysschaert, J.-M.; Narayanaswami, V. Biochemistry 2006, 45, 10947-.de Laureto, P. P.; Tosatto, L.; Frane, E.; Marin, O.; Uversky, V. N.; Fontana, A. Biochemistry 2006, 45, 38, 11523-.Theory 1: C-terminal tail becomes -sheet Theory 2: C-terminal tail rigidified by binding calcium ion

  • 80Val Thr Gly Val Thr Ala Val Ala Gln Lys 90 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala 94 100Ala Thr Gly Phe Val Lys Lys Asp Gln Leu101 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu 113 120Gly Ile Leu Glu Asp Met Pro Val Asp Pro 125 130Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu 136 140Glu Gly Tyr Gln Asp Tyr Glu Pro Glu AlaAcidic ResiduesMutation SitesCa Binding Effective calcium binding has been shown for sequences DXXXD.

  • Seven mutants were expressed:W94/Y1136) W101/Y125 W94/Y1257) W101/Y136W94/Y136W125/Y136W101/Y74Graphic made in PyMol

  • 80Val Thr Gly Val Thr Ala Val Ala Gln Lys 90 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala 94 100Ala Thr Gly Phe Val Lys Lys Asp Gln Leu101 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu 113 120Gly Ile Leu Glu Asp Met Pro Val Asp Pro 125 130Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu 136 140Glu Gly Tyr Gln Asp Tyr Glu Pro Glu AlaAcidic ResiduesMutation SitesCa Binding Effective calcium binding has been shown for sequences DXXXD.Binding sites for Calcium in solution is found between residue 101-113 & residue 125-136

    Nac region/calcium binding