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GNNQQNY Amyloid Fiber: cross-  spine

GNNQQNY Amyloid Fiber: cross-  spine. Nelson, et al. Structure of the cross -spine of amyloid-like fibrils . Nature 435, 773-778 (9 June 2005). Amyloid Unknowns -- Lots!. Universal structure or milieu of structures with a common theme? Mechanism of toxicity?

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GNNQQNY Amyloid Fiber: cross-  spine

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  1. GNNQQNY Amyloid Fiber:cross- spine Nelson, et al. Structure of the cross -spine of amyloid-like fibrils. Nature 435, 773-778 (9 June 2005)

  2. Amyloid Unknowns -- Lots! • Universal structure or milieu of structures with a common theme? • Mechanism of toxicity? • Mechanisms of in vivo control? • Are all amyloids detrimental or are some beneficial? (Lindquist & Kandel) • Species barriers and strains? • Preventatives/drugs?

  3. A1-40 Model by Solid State NMR:double layered -sheet Robert Tycko. Insights into the Amyloid Folding Problem from Solid-State NMR(2003) Biochemistry,42 (11), 3151 -3159,

  4. Model of Ure2p10-39 Yeast Prion:Parallel Superpleated -sheet Kajava, Andrey V. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 7885-7890

  5. A Model by Mutagenesis Williams, et al. Mapping A Amyloid Fibril Secondary Structure Using Scanning Proline Mutagenesis. J. Mol. Biol. 335 (2004): 833-842. Morimoto, et al. Analysis of the secondary structure of -amyloid (A42) fibrils by systematic proline replacement. J. Biol. Chem. 279 (2004): 52781-52788. Masuda, et al. Verification of the turn at positions 22 and 23 of the β-amyloid fibrils with Italian mutation using solid-state NMR. (2005) Bioorganic & Medicinal Chemistry. Article in Press.

  6. Model of Human Prion Protein:Left-handed parallel -helix Govaerts, Ceadric et al. (2004) Proc. Natl. Acad. Sci. USA 101, 8342-8347

  7. The b-helix: A simple b-fold

  8. Examples of Parallel -Helices Spruce Budworm Anti-Freeze Protein (1M8N) Left-handed Chondroitinase B (1DBG) Right-handed T4 Lyzosyme Complex (1K28) Triple Stranded

  9. Left vs. Right-handed -helices Image from: Wetzel, Ronald. Ideas of Order for Amyloid Fibril Structure. (2002) Structure 10: 1031-1036.

  10. 6 homotrimers -helix in p22 Tailspike

  11. Endorhamnosidase Activity From STEINBACHER, et al., PNAS 93:10584–10588, October 1996

  12. Tailspike in vivo folding and aggregation pathways [D] [I] [pT] N SDS-sensitive Soluble SDS-sensitive Soluble -S-S- Tm≈42ºC SDS-sensitive Soluble -S-H Tm=88ºC SDS-resistant Soluble SDS-sensitive Insoluble Nascent Polypeptide Chains [I*] Aggregate

  13. Known Folding Pathway Allows for a Simple Assay of Folding Success N [pT] [I] [D] Nascent Polypeptide Chains [I*] Aggregate

  14. In vivo Folding Characterization • Express chains in cells • Capture conformational states on ice • Lyse cells • Analyze by electrophoresis

  15. Folding Characterization In vivo SDS Gel of Lysates In vitro Native Gel Gel images from Betts and King. Structure (1999) 7:R131-R139

  16. In vivo folding efficiency may be assisted by the ribosome itself • The early folding stages of the newly translated tailspike chain occur in a ribosome associated state Patricia L. Clark & Jonathan King 2001 JBC276:25411

  17. Left vs. Right-handed -helices Image from: Wetzel, Ronald. Ideas of Order for Amyloid Fibril Structure. (2002) Structure 10: 1031-1036.

  18. Hydrophobic Stacks 113 residues identified as participating in b-helix core stacks Ryan Simkovsky

  19. Chondroitinase B Stacking

  20. Threonine Stack Likely Allows for Ice Binding in Anti-Freeze Protein Graether SP, et al. β-Helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect. Nature 406, 325 (2000)

  21. Isolated b-helix (109-544) forms amyloid fibers A) Light Microscope B) Light Microscope C) Electron Microscope D) Congo Red Binding E) Birefringence via cross-polarized light Schuler, Rachel, & Seckler. J. Biol. Chem. (1999) 274:18589-18596.

  22. Tailspike in vivo folding:An Assembly Process [D] [I] [pT] N SDS-sensitive Soluble SDS-sensitive Soluble -S-S- Tm≈42ºC SDS-sensitive Soluble -S-H Tm=88ºC SDS-resistant Soluble SDS-sensitive Insoluble Nascent Polypeptide Chains [I*] Aggregate

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