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Perturbation of the Stability of Amyloid Fibrils through Alteration of Electrostatic Interactions

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 , 2783-2791 (2011) Na Young Kim CHEM 645. Amyloid Fibrils.

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Perturbation of the Stability of Amyloid Fibrils through Alteration of Electrostatic Interactions

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  1. 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, 2783-2791 (2011) Na Young Kim CHEM 645

  2. Amyloid Fibrils • Filamentous, insoluble structures • Misfolding aggregation • Nucleation required • Highly stable once formed • Can be lower energy state than that of native protein • Nanomaterials inspired • Difficult to disaggregate in vivo • Greater than 40 clinical disorders linked to amyloid fibrils • Neurodegenerative Diseases • Alzheimer’s • Parkinson’s • Huntington’s • Type II diabetes Info: Shammas SL et al. Biophys. J.100, 2783-2791 (2011) Nelson R et al., Nature435, 773-778 (2005) Picture: Ross CA, Poirier MA. Nature Medicine10, S10-S17 (2004)

  3. Structure of Amyloid Fibrils Nelson R et al., Nature435, 773-778 (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

  4. Structure of Amyloid Fibrils Nelson R et al., Nature435, 773-778 (2005)

  5. 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

  6. 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

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

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

  9. Structural Changes Below pH 8 Fourier transform infrared spectroscopy (FTIR) amide I’ region (1600-1700 cm-1) used to determine secondary structure content (pH* 1.6-3.6-5.6-7.6)

  10. 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

  11. Kinetics of Dissociation Adjusting pH to 10.6, 11.0, 11.4, and 12.0, and measuring turbidity over time Picture: http://en.wikipedia.org/wiki/Absorbance Equations: http://www.chroma.com/knowledge/introduction-fluorescence/percent-transmission-and-optical-density

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

  13. Kinetics of Dissociation Continued

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

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

  16. 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” www.chem.uwec.edu/Chem455_S05/Pages/.../FTIR_of_proteins.pdf

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