Phospholipid membrane interfaces and metal cations, Ca²⁺ and Cu²⁺, modulate insulin amyloid fibrillation

Metal ions are widely implicated in amyloid fibril (AF) formation, yet their effects have largely been characterized in solution, while the influence of lipid membrane interfaces has received less attention. To address this gap, we herein addressed the question of whether the effects of two divalent cations with contrasting physicochemical properties (Cu²⁺ and Ca²⁺) on AF formation in a model amyloidogenic protein (recombinant human insulin, INS) are the intrinsic properties of these ions in solution or depend on the presence of a lipid membrane interface. AF formation kinetics were monitored using thioflavin T fluorescence, ion accumulation at colloidal surfaces was assessed by zeta potential measurements, and the secondary structural composition of aggregates was characterized by attenuated total reflection Fourier transform infrared spectroscopy and analyzed using singular value decomposition (SVD). In the absence of egg yolk phospholipids small unilamellar vesicles (SUVs), Ca²⁺ accelerated nucleation without altering AF structure, whereas Cu²⁺ inhibited AF nucleation and elongation in a concentration-dependent manner. The introduction of SUVs transformed the direction and magnitude of these effects: Ca²⁺ shifted from being a nucleation promoter to acting as an AF elongation inhibitor, whereas Cu²⁺ inhibition was amplified to near-complete suppression at 100 μM. Zeta potential analysis revealed an apparent depletion of free Cu²⁺ consistent with binding via direct INS coordination below 100 μM-a pattern absent for Ca²⁺-and SVD analysis showed that Cu²⁺ progressively disrupted cross-β structure formation, whereas Ca²⁺ caused no structural change under any condition. These results suggest that the membrane interface does not merely modulate the magnitude of metal ion effects on AF formation but determines their direction. The outcome is governed by the physicochemical identity of each ion: Ca²⁺ acts through nonspecific electrostatic screening and is redirected at the membrane to suppress elongation via interfacial confinement; Cu²⁺ acts through direct protein coordination and is amplified at the membrane by geometric concentration at the two-dimensional interface. The role of metal ions in AF formation cannot be assessed using solution-phase measurements alone, as the membrane interface is an integral determinant of the aggregation outcome.