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?
• Mechanisms of in vivo control?
• Are all amyloids detrimental or are
some beneficial? (Lindquist & Kandel)
• Species barriers and strains?
• Preventatives/drugs?
A1-40 Model by Solid State NMR:
double layered -sheet
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Robert Tycko. Insights into the Amyloid Folding Problem from Solid-State NMR(2003)
Biochemistry, 42 (11), 3151 -3159,
Model of Ure2p10-39 Yeast Prion:
Parallel Superpleated -sheet
Kajava, Andrey V. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 7885-7890
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.
Model of Human Prion Protein:
Left-handed parallel -helix
Govaerts, Ceadric et al. (2004) Proc. Natl. Acad. Sci. USA 101, 8342-8347
The -helix: A simple -fold
Examples of Parallel -Helices
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Spruce Budworm
Anti-Freeze Protein
(1M8N) Left-handed
T4 Lyzosyme Complex
(1K28) Triple Stranded
Chondroitinase B
(1DBG) Right-handed
Left vs.
Right-handed -helices
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image from:
Wetzel, Ronald. Ideas of Order for
Amyloid Fibril Structure. (2002)
Structure 10: 1031-1036.
6 homotrimers
-helix in p22 Tailspike
Endorhamnosidase Activity
From STEINBACHER, et al., PNAS 93:10584–10588, October 1996
Tailspike in vivo folding
and aggregation pathways
[I]
SDS-sensitive
Soluble
[D]
SDS-sensitive
Soluble
Nascent
Polypeptide
Chains
[pT]
-S-STm≈42ºC
SDS-sensitive
Soluble
500 nm
N
-S-H
Tm=88ºC
SDS-resistant
Soluble
SDS-sensitive
Insoluble
Inclusion body of tailspike chains
(nI*) in E. coli cell
[I*]
Aggregate
Known Folding Pathway Allows for a Simple
Assay of Folding Success
N
[I]
[D]
[pT]
Nascent
Polypeptide
Chains
500 nm
[I*]
Inclusion body of tailspike chains
(nI*) in E. coli cell
Aggregate
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
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
In vitro Native Gel
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TIFF (LZW) decompressor
are needed to see this picture.
Gel images from Betts and King. Structure (1999) 7:R131-R139
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
Left vs.
Right-handed -helices
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Image from:
Wetzel, Ronald. Ideas of Order for
Amyloid Fibril Structure. (2002)
Structure 10: 1031-1036.
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
QuickTime™ and a
TIFF (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
Light Microscope
amyloid fibers A)
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.
Tailspike in vivo folding:
An Assembly Process
[I]
SDS-sensitive
Soluble
[D]
SDS-sensitive
Soluble
Nascent
Polypeptide
Chains
[pT]
-S-STm≈42ºC
SDS-sensitive
Soluble
500 nm
N
-S-H
Tm=88ºC
SDS-resistant
Soluble
SDS-sensitive
Insoluble
Inclusion body of tailspike chains
(nI*) in E. coli cell
[I*]
Aggregate