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

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.

Polymorphic Fibrillation of the Destabilized Fourth Fasciclin-1 Domain Mutant A546T of the Transforming Growth Factor-β-induced Protein (TGFBIp) Occurs through Multiple Pathways with Different Oligomeric Intermediates

“…For FAS1-4 A546T, polymorphism is induced by differences in protein concentration. Fibrillation of the peptide hormone glucagon at different peptide concentrations also induces fibril polymorphism (67). This polymorphism arises from a concentration-dependent equilibrium between the monomeric form and a trimer.…”

Novel Mechanistic Insight into the Molecular Basis of Amyloid Polymorphism and Secondary Nucleation during Amyloid Formation

“…We therefore sought to investigate this process and to elucidate the molecular factors governing its occurrence and specificity. Previous studies by Andersen et al 20,56 and others [59][60][61] demonstrated that parts of fibril surfaces have the capacity to nucleate fibrillization resulting in the branching, growth away or along the parent fibril. Under conditions that favored the formation of other fibril varieties (e.g., type-4 and type-5), no secondary-nucleation events were observed, even after the addition of fresh Aβ42-M (Fig.…”

“…9,10 Previous studies involving in situ atomic force microscopy (AFM) or transmission electron microscopy (TEM) have revealed Aβ42 fibrillogenesis to proceed via the formation of highmolecular-weight protofibrils. 11,12 The kinetics of protofibril formation and stability and the structure of the final fibrils depend greatly upon ambient conditions such as pH, temperature, buffer composition, and peptide concentration, [13][14][15][16][17][18][19][20] as well as upon the hydrophobicity or hydrophilicity of the substrate. 21 Although the mechanism underlying the aggregation of Aβ peptides in vitro has been the subject of numerous investigations over the past two decades, many fundamental questions remain unanswered.…”

ThT 101: a primer on the use of thioflavin T to investigate amyloid formation

“…Fibril formation is accompanied by changes in secondary structure. The native state of amyloidogenic precursor proteins can be highly diverse, ranging from purely a-helical (myoglobin [114]), mixtures of a-helices and b-sheets (insulin [115] and lysozyme [116]), a/b-mixed structures (HypF N-terminal domain [117]), b-barrels (Ig light chain [118]) or natively unfolded (a-synuclein [119] and glucagon [120]). Upon fibril formation, some proteins undergo first a decrease in ordered secondary structure and a concomitant increase in random coil structures, followed by a gain in b-sheet-rich features, which make up the backbone of amyloid fibrils.…”