Molecular Details of A Salt-Bridge and Its Role in Insulin Fibrillation by NMR and Raman Spectroscopic Analysis

Insulin, a simple polypeptide hormone with huge biological importance, has long been known to self-assemble in vitro and form amyloid like fibrillar aggregates. Utilizing high-resolution NMR, Raman spectroscopy and computational analysis, we demonstrate that the fluctuation of the carboxyl terminus (C-ter) residues of the insulin B-chain plays a key role in the growth phase of insulin aggregation. By comparing insulin sourced from bovine (BI), human (HI) and the modified glargine (GI), we observed reduced aggregation propensity in the GI variant resulting from two additional Arg residues at its C-ter. NMR analysis showed atomic contacts and residue specific interactions, particularly the salt-bridge and H-bond formed among C-ter residues Tyr26B, Lys29B and Glu4A. These inter-residue interactions were reflected in strong NOEs among Arg31BδH-Glu4AδH, and Lys29BδHs-Glu4AδH in GI, as well as the associated downfield chemical shift of several A-chain amino terminus (N-ter) residues. The two additional Arg resi...