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Sequence control of Aggregation

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 a 1-Antitrypsin. Carrell, et al. (1998) Curr. Opin. Struct. Biol. 8:799-809. Inactive

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Sequence control of Aggregation

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  1. Sequence control of Aggregation Some Examples

  2. Domain Swapping

  3. Domain Swapping of RNase A Yanshun & Eisenberg (2002) Protein Sci. 6:1285-1299.

  4. Loop-Sheet Insertion

  5. Loop-sheet Insertion of a1-Antitrypsin Carrell, et al. (1998) Curr. Opin. Struct. Biol. 8:799-809. Inactive (Stable) Loop-sheet Insert Polymer Active (Metastable)

  6. 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

  7. Human D-crystallin Fluorescence emission spectra Trp68 Trp156 Trp42 Trp130

  8. Equilibrium Unfolding/Refolding in GdnHCl at 37oC Refolding Unfolding

  9. Folding pathway of HD-Crys

  10. Refolding: AFM Time Course 300 nm 1 mm 1 mm Native 5 minutes 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

  11. N-terminal Met43 Phe56 Ile81 C-terminal Leu144 Val131 Val169 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%

  12. Equilibrium unfolding/refolding F56A V131A

  13. Measure Solution Turbidity at 350 nm Unfold at 2.3 M GuHCl Dilute to 0.23-2.3 M GuHCl Refolded to 0.23 M GuHCl Native control Refolding from intermediate 10 g/ml protein 100 g/ml protein

  14. Model for Crystallin Unfolding, Damage and Cataract Formation Within the Lens

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