Sequence control of Aggregation

1
Sequence control of Aggregation

• Some Examples

2
Domain Swapping
3
Domain Swapping of RNase A
Yanshun Eisenberg (2002) Protein Sci.
61285-1299.
4
Loop-Sheet Insertion
5
Loop-sheet Insertion of a1-Antitrypsin
Carrell, et al. (1998) Curr. Opin. Struct. Biol.
8799-809.
Inactive (Stable)
Loop-sheet Insert
Polymer
Active (Metastable)
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The ubiquitous crystallins

• - large polydisperse complexes, molecular
  chaperone activity
• ? - oligomeric, two domain structure, ?B2
  dimerizes by domain swapping
• ? - monomeric, similar domain structure as
• ?-crystallins

Slingsby and Jaenicke (1999) Eye, Pt 3b 395-402
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Human ?D-crystallin
Fluorescence emission spectra
Trp68
Trp156
Trp42
Trp130
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Equilibrium Unfolding/Refolding in GdnHCl at 37oC
Refolding
Unfolding
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Folding pathway of H?D-Crys
10
Refolding AFM Time Course
300 nm
1 mm
1 mm
5 minutes
Native
30 seconds
300 nm
1 mm
1 mm
1 mm
1 mm
1 mm
24 minutes
54 minutes
54 minutes
1 mm
1 mm
1 mm
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Hydrophobic domain interface residues
N-terminal domain
C-terminal domain
Conservation among 35 ?-crystallin sequences
Phe56 - Phe 80, Val 8.5, Ile 8.5, Leu
3 Val131 - Val 54.3, Ile 28.5, Leu
17.2 Ile81 - Ile 80, Val 8.5, Leu 5.5,
Pro 3, Thr 3 Leu144 - Leu 68.5, Tyr 20,
Phe 11.5 Met43 - Met 77, Val 11.5, Ala
8.5, Ile 3 Val169 - Val 49, Ile 42, Ala
3, Met 3, Leu 3
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Equilibrium unfolding/refolding
F56A
V131A
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Refolding from intermediate
10 ?g/ml protein
100 ?g/ml protein
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Model for Crystallin Unfolding, Damage and
Cataract Formation Within the Lens