pH Dependence of Amylin Fibrillization

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic beta-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the alpha-amino group and His18. Our approach to measuring the pK(a) values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The alpha-amino group at the unstructured N-terminus of amylin has a pK(a) near 8.0, similar to the value in random coil models. By contrast, His 18, which is involved in the intermolecular beta-sheet structure of the fibrils, has a pK(a) that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered pK(a) of His 18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic beta-cells. In addition to the two expected pK(a) values, we detected an apparent pK(a) of similar to 4.0 for the amylin-derived peptide NAc-SNNFGAILSS-NH2, which has no titratable groups. This pK(a) is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein.