GNNQQNY Amyloid Fiber: cross- spine

Nelson, et al. Structure of the cross -spine of amyloid-like fibrils.Nature 435, 773-778 (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), 3151 -3159,

A1-40 Model by Solid State NMR:double layered -sheet

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Kajava, Andrey V. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 7885-7890

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): 833-842.

Morimoto, et al. Analysis of the secondary structure of -amyloid (A42) 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.

Govaerts, Ceadric et al. (2004) Proc. Natl. Acad. Sci. USA 101, 8342-8347

Model of Human Prion Protein:Left-handed parallel -helix

The -helix: A simple -fold

Examples of Parallel -Helices

Chondroitinase B(1DBG) Right-handed

Spruce BudwormAnti-Freeze

Protein (1M8N) Left-

handed

QuickTime™ and aTIFF (LZW) decompressor


T4 Lyzosyme Complex(1K28) Triple

Stranded



Image from:Wetzel, Ronald. Ideas of Order for Amyloid Fibril Structure. (2002) Structure 10: 1031-1036.

Left vs. Right-handed -helices

-helix in p22 Tailspike6 homotrimers

Endorhamnosidase Activity

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

Tailspike in vivo folding and aggregation pathways

Inclusion body of tailspike chains(nI*) in E. coli cell

500 nmNascent Polypeptide

Chains

[I] [D] [pT] N

Aggregate[I*]

-S-HTm=88ºC

SDS-resistantSoluble

-S-S-Tm≈42ºC

SDS-sensitiveSoluble



SDS-sensitiveInsoluble

Known Folding Pathway Allows for a Simple Assay of Folding

Success



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: 1031-1036.

Left vs. Right-handed -helices

Hydrophobic Stacks113 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 (109-544) forms amyloid fibers A) Light

MicroscopeB) Light MicroscopeC) Electron MicroscopeD) Congo Red BindingE) Birefringence via cross-polarized light

Schuler, Rachel, & Seckler. J. Biol. Chem. (1999) 274:18589-18596.

Tailspike in vivo folding:An Assembly Process



Chains

[I] [D] [pT] N

Aggregate[I*]

-S-HTm=88ºC

SDS-resistantSoluble

-S-S-Tm≈42ºC




SDS-sensitiveInsoluble

GNNQQNY Amyloid Fiber: cross- spine

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