0. Biophysical Journal 100, 2783-2791 (2011)
Perturbation of the Stability of Amyloid Fibrils through Alteration of Electrostatic Interactions
Shammas SL, Knowles TPJ, Baldwin AJ, MacPhee CE, Welland ME, Dobson CM, and Devlin GL
Biophysical Journal 100, (2011)
Na Young Kim
CHEM 645

1. Amyloid Fibrils Filamentous, insoluble structures
Greater than 40 clinical disorders linked to amyloid fibrils
Misfolding  aggregation
Nucleation required
Neurodegenerative Diseases
Highly stable once formed
Alzheimer’s
Can be lower energy state than that of native protein
Parkinson’s
Huntington’s
Nanomaterials inspired
Type II diabetes
Difficult to disaggregate in vivo
Info: Shammas SL et al. Biophys. J. 100, (2011)
Nelson R et al., Nature 435, (2005)
Picture: Ross CA, Poirier MA. Nature Medicine 10, S10-S17 (2004)

2. Structure of Amyloid Fibrils
Nelson R et al., Nature 435, (2005)
Common characteristics
Elongated, unbranched
Cross-beta diffraction pattern
Binding Congo red and thioflavin T
Unusual stability
3 levels of organization within fibril
Formation of beta-sheet (H-bonds within each sheet)
Pair-of-sheets (van der Waals forces; dry interface; “steric zipper”)
Non-covalent forces form fibrils

3. Structure of Amyloid Fibrils
Nelson R et al., Nature 435, (2005)

4. Insulin Readily forms amyloid fibrils at acidic pH
These dissociate under alkaline conditions, but by what mechanism?
Dissociation due to charge change at higher pH
Effects of partial distruption of beta-sheet on dissociation via denaturant addition
Kinetics of dissociation
Bovine Insulin (Hexameric form) PDB: 2ZP6

5. Dissociation of Amyloid Fibrils at High pH
Amyloid fibrils formed at pH 2.0 by seeding
Incubated amyloid fibrils at room temperature and various pH for 48h

6. Driving Force Behind Dissociation?
These simple electrostatic considerations accurately described the dissociation of these charged fibrils

7. Is Nothing Happening Below pH 8?
Structural changes apparent above pH 4, but no dissociation of insoluble fibrils until above pH 8 (from TEM)

8. Structural Changes Below pH 8
Fourier transform infrared spectroscopy (FTIR) amide I’ region ( cm-1) used to determine secondary structure content (pH* )

9. Beta-sheet Content Correlates Well to Fibril Stability
GdnSCN dissociation at pH 2, 4, 6, and 8
Fit to linear polymerization model to find fibril stabilities
Fibril stabilities correlated well to beta-sheet relative intensity pH
deltaG (kJ/mol) 2
/- 1.2 4
/- 1.6 6
/- 0.9 8
/- 0.6

10. Kinetics of Dissociation
Adjusting pH to 10.6, 11.0, 11.4, and 12.0, and measuring turbidity over time
Picture:
Equations:

11. Kinetics of Dissociation Continued
After fitting double-exponential model to OD versus time data Temperature dependence showed Arrhenius-type behavior

12. Kinetics of Dissociation Continued

13. Conclusions pH < 4 pH 4 – 8 pH > 8
Highly stable insulin amyloid fibrils
Decrease in beta-sheet network
Increased electrostatic repulsions due to large net charge
Decrease in thermodynamic stability

14. Significance
Significance of beta-sheet content on thermodynamic stability of amyloid fibrils
“Fibrillar state can be thermodynamically more stable than the native state”

15. Questions?

17. Outline
“Fibril stability is susceptible to electrostatic repulsion between constituent polypeptide chains” (pH 2 to 12)
“Thermodynamic stability of fibrils is reduced by partial disruption of beta-sheet structure” (pH 4 to 8)
“The kinetics of insulin fibril dissociation is dependent on the ionization state of a single side chain” (pH>10)

18. Explanation of Eq 2 Assumptions:
“Each monomer in the fibril experiences a repulsive elctrostatic interaction with its nearest neighbors”
Dynamic equilibrium exists between the fibrillar and soluble states of insulin
This can be described by classical linear polymerization theory

19. FTIR of Proteins
Gallagher, W. “FTIR Analysis of Protein Structure”