Stabilization of α-Helical Folded Structures Retards Hydrophobic Zipping and Fibrillation of Bovine Insulin: A Key...

Insulin is an α-helical-rich globular protein that is well-stabilized via several noncovalent forces including the inter-residue/intersubunit hydrophobic interactions. However, similar noncovalent forces, although of different degrees and orientations, effectuate many proteins to assemble and adapt thermodynamically stable β-sheet-rich fibrillar aggregates, causing a severe impact on their native structure and function. This fibrillation of proteins involves a key event, which is the zipping of hydrophobic amyloidogenic regions that are exposed intrinsically or become bared in the folded proteins under harsh conditions. This study has revealed that Coomassie Brilliant Blue G-250 (CBBG) can inhibit the essential zipping processes and stabilize the α-helical structure of bovine insulin (BI), resulting in a significant delay in the fibril formation. The interaction of CBBG with BI was found to be a thermodynamically favorable event, with it being an enthalpy-driven process (ΔH⁰ -88.04 kcal/mol), with the change in Gibb's free energy (ΔG⁰) observed to be ∼ -6.98 kcal/mol. Surface-enhanced Raman scattering measurements showed a characteristic α-helical signal of the protein at 1649 cm^-1 in the presence of CBBG, suggesting the enhanced thermal stability of the hormone. Computational analysis further revealed that CBBG binds to both chains A and B of bovine insulin and boosts the folding stability in the monomeric state, causing a significant reduction in its structural fluctuation. The sulfonate moieties of CBBG showed significant intermolecular interactions with the B chain of N-terminal segments. Specifically, one sulfonate group formed multiple hydrogen bonds with both the backbone amide group and the terminal amine. Also, the N-terminal phenylalanine residue of BI (F1B) was found to have a significant contribution to the hydrophobic π-π stacking interactions with the CBBG aromatic phenyl ring.