Elucidating the Role of Oligomers in Insulin Aggregation using Biophysical Methods

Abstract

Protein misfolding and aberrant fibrillization underlie many neurodegenerative conditions, such as Alzheimer's and Parkinson's disease. Insulin, which is composed of two covalently bonded peptide chains, exists in vivo mostly in a native hexameric state but becomes amyloidogenic under certain conditions: at high temperature with neutral pH (7.4) and agitation or with low pH (1.6) and quiescence. To investigate the mechanisms that drive insulin aggregation, we monitor its self-assembly into fibrils by kinetic fluorescence spectroscopy, which uses Thioflavin T (ThT), a fluorescent dye that binds to the cross-β structure of amyloid fibrils. At low pH, insulin behaves similarly to other amyloid proteins; kinetic rate of fibrillization increases with concentration. At neutral pH, we observe an increase of the kinetic rate of fibrillization with low insulin concentration (2.5 -- 25 μM), whereas at higher concentrations (25 -- 100 μM) the opposite trend is observed. To explain this observation, we utilize photo induced cross-linking of unmodified proteins (PICUP) and Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) to determine the oligomeric population of pre-fibrillar stages of insulin self-assembly. Preliminary results show a shift toward larger oligomers at insulin concentrations in the vicinity of 25 μM. As self-assembly advances and fibrils start to form (as observed by ThT fluorescence), PICUP/SDS-PAGE shows progressively decreased oligomer abundances. Insulin aggregation is also monitored via atomic force microscopy (AFM) to investigate differences in morphology between the two methods used to induce aggregation and the corresponding time evolution of oligomeric species. Our results are consistent with oligomer formation that is on the pathway to fibril formation, thereby elucidating a key interplay between oligomers and fibrils in insulin aggregation.