Deciphering the role of chondroitin sulfate a in insulin fibrillation at room temperature: a biophysical and computational perspective.

Chondroitin sulfate A (CSA), a natural mucopolysaccharide of the extracellular matrix, plays a critical role in amyloidogenesis. This study explores the interaction between CSA and human insulin, with emphasis on conformational transitions and amyloid fibril formation. A combination of biophysical (UV-Vis spectroscopy, Far-UV CD, light scattering, ThT fluorescence), microscopic (TEM imaging), and computational approaches was employed to assess CSA's impact on insulin solubility, aggregation propensity, and structural rearrangements. Our findings reveal that CSA-induced amyloid formation proceeds through concentration-dependent aggregation of insulin. CSA exposure triggered dose-dependent aggregation of insulin, reaching saturation at a defined concentration. Light scattering increased linearly below 0.125 mg/mL CSA and plateaued thereafter. Far-UV CD analysis demonstrated a secondary structural transition from α-helical to cross-β-sheet above 0.125 mg/mL CSA, a hallmark of amyloid fibrils. ThT fluorescence kinetics and TEM imaging confirmed the presence of amyloid-like structures in CSA-treated insulin samples. Molecular docking identified a stable CSA binding site on the insulin surface mediated by hydrogen bonding and van der Waals interactions. Complementary molecular dynamics simulations suggest a possible structural basis by which CSA may influence insulin aggregation, demonstrating stable CSA binding and pH-dependent conformational changes in insulin over 200 ns. Collectively, these results establish CSA as an aggregation-promoting cofactor and highlight the utility of molecular simulations in dissecting amyloidogenic transitions. This work advances mechanistic understanding of glycosaminoglycan-mediated protein aggregation and offers insights relevant to pharmaceutical formulations and amyloid research.