Sequence control of Aggregation

Some Examples

Domain Swapping

Domain Swapping of RNase A

Yanshun & Eisenberg (2002) Protein Sci. 6:1285-1299.

Loop-Sheet Insertion

Loop-sheet Insertion of 1-Antitrypsin

Active (Metastable)

Inactive(Stable)

Loop-sheetInsert

Polymer

Carrell, et al. (1998) Curr. Opin. Struct. Biol. 8:799-809.

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

Human D-crystallinFluorescence emission spectra

Trp68

Trp42

Trp156

Trp130

Equilibrium Unfolding/Refolding in GdnHCl at 37oC

Refolding

Unfolding

Folding pathway of HD-Crys

Refolding: AFM Time Course

5 minutes30 seconds

24 minutes 54 minutes 54 minutes

Native300 nm

300 nm

1 m

1 m 1 m

1 m1 m

1 m 1 m1 m

1 m 1 m

Hydrophobic domain interface residues

N-terminal domain C-terminal domain

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%

Conservation among 35 -crystallin sequences:

N-terminal

Met43Phe56Ile81

C-terminal

Leu144Val131Val169

Equilibrium unfolding/refolding

F56A V131A

Unfold at 2.3 M GuHCl Dilute to 0.23-2.3 M GuHCl

Measure Solution Turbidity at 350

nm

Native control Refolded to 0.23 M GuHCl

10 g/ml protein 100 g/ml protein

Refolding from intermediate

Model for Crystallin Unfolding, Damage and Cataract Formation

Within the Lens