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

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?

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

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

A  Model by Mutagenesis Williams, et al. Mapping A  Amyloid Fibril Secondary Structure Using Scanning Proline Mutagenesis. J. Mol. Biol. 335 (2004): Morimoto, et al. Analysis of the secondary structure of  -amyloid (A  42) fibrils by systematic proline replacement. J. Biol. Chem. 279 (2004): 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.

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

The  -helix: A simple  -fold

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

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

 -helix in p22 Tailspike 6 homotrimers

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

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

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

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

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

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 JBC 276:25411

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

Hydrophobic Stacks 113 residues identified as participating in  -helix core stacks Ryan Simkovsky

Chondroitinase B Stacking

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)

Isolated  -helix ( ) 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:

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