Structural Insight of Amyloidogenic Intermediates of Human Insulin

Engaging Raman spectroscopy as a primary tool, we investigated the early events of insulin fibrilization and determined the structural content present in oligomer and protofibrils that are formed as intermediates in the fibril formation pathway. Insulin oligomer, as obtained upon incubation of zinc-free insulin at 60 °C, was mostly spherical in shape, with a diameter of 3–5 nm. Longer incubation produced “necklace”-like beaded protofibrillar assembly species. These intermediates eventually transformed into 5–8 nm thick fibers with smooth surface texture. A broad amide I band in the Raman spectrum of insulin monomer appeared at 1659 cm^–1, with a shoulder band at 1676 cm^–1. This signature suggested the presence of major helical and extended secondary structure of the protein backbone. In the oligomeric state, the protein maintained its helical imprint (∼50%) and no substantial increment of the compact cross-β-sheet structure was observed. A nonamide helix signature band at 940 cm^–1 was present in the oligomeric state, and it was weakened in the fibrillar structure. The 1-anilino-8-naphthalene-sulfonate binding study strongly suggested that a collapse in the tertiary structure, not the major secondary structural realignment, was the dominant factor in the formation of oligomers. In the fibrillar state, the contents of helical and disordered secondary structures decreased significantly and the β-sheet amount increased to ∼62%. The narrow amide I Raman band at 1674 cm^–1 in the fibrillar state connoted the formation of vibrationally restricted highly organized β-sheet structure with quaternary realignment into steric-zipped species