No abstract

Using the peptide hormone glucagon and Abeta(1-40) as model systems, we have sought to elucidate the mechanisms by which fibrils grow and multiply. We here present real-time observations of growing fibrils at a single-fibril level. Growing from preformed seeds, glucagon fibrils were able to generate new fibril ends by continuously branching into new fibrils. To our knowledge, this is the first time amyloid fibril branching has been observed in real-time. Glucagon fibrils formed by branching always grew in the forward direction of the parent fibril with a preferred angle of 35-40 degrees . Furthermore, branching never occurred at the tip of the parent fibril. In contrast, in a previous study by some of us, Abeta(1-40) fibrils grew exclusively by elongation of preformed seeds. Fibrillation kinetics in bulk solution were characterized by light scattering. A growth process with branching, or other processes that generate new ends from existing fibrils, should theoretically give rise to different fibrillation kinetics than growth without such a process. We show that the effect of adding seeds should be particularly different in the two cases. Our light-scattering data on glucagon and Abeta(1-40) confirm this theoretical prediction, demonstrating the central role of fibril-dependent nucleation in amyloid fibril growth.

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.