Engineered diselenide bridge protects insulin glargine from fibrillation and enhances stability

Polypeptide cross-β assembly, or fibrillation, is a common degradation pathway for peptide and protein therapeutics, limiting their shelf life and requiring complex "cold chain" storage. Insulin glargine, a long-acting insulin analog, is particularly susceptible to aggregation at elevated temperatures because its acidic formulation prevents protective zinc-mediated hexamer assembly, unlike wild-type insulin. This instability impairs activity and poses significant logistical challenges for global distribution and patient use, highlighting a critical need for enhanced protein stability.

Researchers engineered a diselenide bridge into insulin glargine by replacing CysA6 and CysA11 with selenocysteine residues. They then compared the biophysical properties of this modified analog against wild-type insulin and unmodified insulin glargine. Key assays included measuring fibrillation lag time at 37°C, assessing resistance to pepsin cleavage, guanidine denaturation, and thermal unfolding. NMR spectroscopy and 1H-2H amide-proton exchange were used to characterize conformational dynamics, complemented by molecular dynamics simulations.

At 37°C, unmodified insulin glargine exhibited a fourfold accelerated fibrillation lag time compared to wild-type insulin, indicating its inherent instability. This instability is a significant concern for its real-world application. The engineered diselenide bridge in the insulin glargine analog completely circumvented this accelerated fibrillation, demonstrating significant protection against aggregation. This enhanced stability correlated with augmented resistance to pepsin cleavage, guanidine denaturation, and thermal unfolding. While NMR structures of the analog and native glargine were similar, detailed 1H-NMR chemical shifts, helix-associated NOEs, and amide-resonance line widths provided evidence of damped conformational fluctuations in the diselenide-bridged analog. Further, 1H-2H amide-proton exchange patterns confirmed reduced dynamics, a finding supported by molecular dynamics simulations, suggesting a direct link between native-state conformational fluctuations and fibrillation propensity. This "dynamic engineering" approach offers a robust strategy for enhancing protein stability.