Selection and Characterization of Non‐Amyloidogenic Mutants of the Type II Diabetes Linked Protein, IAPP

The 37‐residue polypeptide IAPP is known to aggregate into insoluble amyloid or small oligomers. Both of these oligomeric states appear to play a role in the death of pancreatic â‐islet cells in type II diabetes. While IAPP has been known to be the primary component of type II diabetes amyloid, the molecular interactions responsible for this aggregation have not been identified. To identify the aggregation‐prone region(s), we constructed a library of randomly generated point mutants of IAPP. This mutant IAPP library was expressed in E. coli as genetic fusions to the reporter protein enhanced green fluorescent protein (EGFP). Because IAPP aggregates rapidly, both independently and when fused to EGFP, the fusion protein does not yield a functional, fluorescent EGFP. However, mutations of IAPP that result in non‐amyloidogenic sequences remain soluble and allow EGFP to fold and fluoresce. Using this screen, we identified 22 single mutations, 4 double mutations and 2 triple mutations of IAPP that appear to be less amyloidogenic than wild type human IAPP. Several selected variants were chemically synthesized and their amyloidogenic properties analyzed with Thioflavin T binding and atomic force microscopy measurements. A comparison of these sequences suggests an additional aggregation‐prone region outside of the 20–29 region of IAPP.