Glucagon Amyloid-like Fibril Morphology Is Selected via Morphology-Dependent Growth Inhibition

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Abstract
The 29-residue peptide hormone glucagon readily fibrillates at low pH, but the structure and morphology of the fibrils are very sensitive to the environmental conditions. Here we have investigated the mechanism behind the differences in morphology observed when glucagon fibrils are formed at different peptide concentrations. Electron microscopy shows that fibrils formed at low glucagon concentration (0.25 mg/mL) are twisted, while fibrils formed at high concentration (8 mg/mL) are straight. Monitoring the fibrillation kinetics at different concentrations, we find that the lag time has an unexpected maximum at a concentration of 1 mg/mL, with faster fibrillation at both lower and higher concentrations. Seeding experiments show that small amounts of straight fibril seeds can accelerate fibril growth at both low and high glucagon concentration, while twisted fibril seeds cannot grow at high concentrations. We conclude that there exists a morphology-dependent mechanism for inhibition of glucagon fibril growth. Light scattering experiments indicate that glucagon is mainly monomeric below 1 mg/mL and increasingly trimeric above this concentration. We propose that the glucagon trimer is able to specifically inhibit growth of the twisted fibril morphology. Such inhibitory binding of molecules in an unproductive conformation could also play a role in the selection of morphologies for other fibril-forming peptides and proteins.