RESUMEN
Insulin fibrillation provides a model for a broad class of amyloidogenic diseases. Conformational distortion of the native monomer leads to aggregation-coupled misfolding. Whereas beta-cells are protected from proteotoxicity by hexamer assembly, fibrillation limits the storage and use of insulin at elevated temperatures. Here, we have investigated conformational distortions of an engineered insulin monomer in relation to the structure of an insulin fibril. Anomalous (13)C NMR chemical shifts and rapid (15)N-detected (1)H-(2)H amide-proton exchange were observed in one of the three classical alpha-helices (residues A1-A8) of the hormone, suggesting a conformational equilibrium between locally folded and unfolded A-chain segments. Whereas hexamer assembly resolves these anomalies in accordance with its protective role, solid-state (13)C NMR studies suggest that the A-chain segment participates in a fibril-specific beta-sheet. Accordingly, we investigated whether helicogenic substitutions in the A1-A8 segment might delay fibrillation. Simultaneous substitution of three beta-branched residues (Ile(A2) --> Leu, Val(A3) --> Leu, and Thr(A8) --> His) yielded an analog with reduced thermodynamic stability but marked resistance to fibrillation. Whereas amide-proton exchange in the A1-A8 segment remained rapid, (13)Calpha chemical shifts exhibited a more helical pattern. This analog is essentially without activity, however, as Ile(A2) and Val(A3) define conserved receptor contacts. To obtain active analogs, substitutions were restricted to A8. These analogs exhibit high receptor-binding affinity; representative potency in a rodent model of diabetes mellitus was similar to wild-type insulin. Although (13)Calpha chemical shifts remain anomalous, significant protection from fibrillation is retained. Together, our studies define an "Achilles' heel" in a globular protein whose repair may enhance the stability of pharmaceutical formulations and broaden their therapeutic deployment in the developing world.