Post on 28-Dec-2015
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
Examples of Parallel -Helices
Chondroitinase B(1DBG) Right-handed
Spruce BudwormAnti-Freeze
Protein (1M8N) Left-
handed
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
T4 Lyzosyme Complex(1K28) Triple
Stranded
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Image from:Wetzel, Ronald. Ideas of Order for Amyloid Fibril Structure. (2002) Structure 10: 1031-1036.
Left vs. Right-handed -helices
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-sensitiveSoluble
SDS-sensitiveSoluble
SDS-sensitiveInsoluble
Known Folding Pathway Allows for a Simple Assay of Folding
Success
Inclusion body of tailspike chains(nI*) in E. coli cell
500 nmNascent 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
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
In vivo SDS Gel of Lysates
Gel images from Betts and King. Structure (1999) 7:R131-R139
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
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
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Image from:Wetzel, Ronald. Ideas of Order for Amyloid Fibril Structure. (2002) Structure 10: 1031-1036.
Left vs. Right-handed -helices
Threonine Stack Likely Allows for Ice Binding in Anti-Freeze Protein
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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.