The 29-residue peptide hormone glucagon forms many different morphological types of amyloid-like fibrils, depending on solvent conditions. Here, we combine time-series far-UV CD with singular value decomposition analysis to reveal six different conformational states populated during fibrillation at 25 °C and pH 2.5. The existence of these states is supported by complementary fluorescence and electron microscopy data. This highlights a multitude of structural transitions of glucagon from unordered structure to β sheets, β turns and further tertiary-level changes. We attribute the observed unusual far-UV CD spectra to tertiary-level structural changes during the formation and maturation of fibrils. The fibrillation model for the whole process involves the formation of three oligomeric species and two different morphologies of fibrils in the same solution. The visualization of annular pore-like species in the early stages of glucagon fibrillation and the prevalence of such species in the amyloidogenesis of several proteins indicates that they may be a common feature of the fibrillation process. This study gives significant insights into the stepwise conversion of soluble glucagon to its fibrillar state and identifies the importance of fibril twisting for its thermodynamic stabilization.
Combining time-series far UV circular dichroism spectra with singular value decomposition reveals six different conformational states populated during fibrillation of glucagon. In combination with fluorescence and electron microscopy data, we propose a fibrillation model which involves formation of three oligomeric species (including an annular pore-like species) and two different morphologies of fibrils (twisted and straight) in the same solution.