The effects of various membrane physical-chemical properties on the aggregation kinetics of insulin.

In a simplified approach to the in vivo situation, where pathogenic fibrillar protein deposits are often found associated with cellular membranes, the aggregation kinetics of insulin in the presence of various model biomembranes were investigated using the Thioflavin T (ThT) fluorescence assay. The lipid dynamics near the gel-fluid transition, the chain length of saturated lipids and the presence of DOPE or DOPS in DOPC-vesicles modulate the aggregation kinetics of insulin in an indifferent, an aggregation-accelerating or an aggregation-inhibiting manner, subtly depending on the pH-value and the presence of salt. The rate of insulin aggregation in bulk solution dominates the overall aggregation process in most cases at low pH, where the lipid additives exert no effect on the aggregation kinetics. The occurrence of dynamic line defects near the gel-fluid transition temperature of DSPC facilitates a partial membrane insertion of the protein, which in turn shields exposed hydrophobic protein patches from intermolecular association and hence inhibit aggregation. An exclusively aggregation-accelerating effect was observed in the presence of 0.1M NaCl for all lipid additives investigated, which is likely due to an enhanced surface accumulation of the protein. Apart from weak dipole-dipole, dipole-monopole and hydrogen bonding interactions, the release of curvature elastic stress in mixed DOPC/DOPE-membranes and preferred interactions of insulin with carboxylic groups in DOPC/DOPS-membranes favour an increased surface accumulation. At neutral pH, a partial insertion of insulin into the lipid bilayer is favoured, which accounts for the aggregation-inhibiting effect of all lipid bilayer systems studied except those containing DOPS. Generally, the extent of inhibition increases with the lipid chain length and the extent of curvature stress in mixed unsaturated lipid membranes and also when the gel-fluid transition temperature of pure phospholipids is approached. The accelerating effect of DOPS on the aggregation of insulin under net electrostatic repulsion at pH 7.4 remains to be elucidated, yet, it might result from increased surface accumulation and/or faster/more extensive unfolding of the protein without a subsequent membrane insertion. These results demonstrate that a delicate interplay between different physical and chemical properties of lipid membranes has to be taken into account in a detailed discussion of membrane-associated protein aggregation phenomena.