Interfacial sequestration by phospholipid membranes and buffer-dependent dual pathways in heat-induced insulin...

Protein aggregation at biological interfaces is a central problem in colloid and surface science. However, the mechanisms by which biological membranes modulate protein aggregation under thermal and shear stresses remain poorly defined. Here, we show that the heat-induced aggregation of recombinant human insulin (0.25 mg/mL) at 338 K proceeds through two competing pathways, bulk-phase nucleation and membrane-assisted nucleation, with phospholipid bilayers acting as catalytic scaffolds. Using Thioflavin T (ThT) fluorogenic assays, kinetic modeling, and buffer-dependent modulation of ionic strength, we found that egg yolk phosphatidylcholine small unilamellar vesicles (SUVs; mean diameter, 100 nm) accelerated early nucleation while suppressing the accumulation of ThT detectable fibrils. Lipid removal experiments revealed that this attenuation arises from transient sequestration or interfacial masking of fibrils rather than inhibition or degradation and that the total fibril mass is greater in the presence of SUVs. Comparative studies of inorganic phosphate (10 mM phosphate/NaOH, pH 5.5-8.0) and organic amine buffers (5 mM bis-tris/5 mM tricine/HCl, pH 5.5-8.0) show that ionic composition tunes the balance between bulk-phase and interfacial nucleation by altering electrostatic screening and membrane-protein interactions. The principles revealed here may be extended to other amyloidogenic proteins whose aggregation is shaped by membrane interactions.