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

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, 2783-2791 (2011) Nelson R et al., Nature 435, 773-778 (2005) Picture: Ross CA, Poirier MA. Nature Medicine 10, S10-S17 (2004)

Structure of Amyloid Fibrils Nelson R et al., Nature 435, 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

Structure of Amyloid Fibrils Nelson R et al., Nature 435, 773-778 (2005)

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

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

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

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

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)

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 -56.3 +/- 1.2 4 -55.4 +/- 1.6 6 -43.2 +/- 0.9 8 -33.3 +/- 0.6

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

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

Kinetics of Dissociation Continued

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

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

Questions?

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)

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

FTIR of Proteins Gallagher, W. “FTIR Analysis of Protein Structure” www.chem.uwec.edu/Chem455_S05/Pages/.../FTIR_of_proteins.pdf