PMC

Schematic representation of the distance between a charged residue at one particular position in a parallel and an antiparallel amyloid β-sheet.

Examples of some of the different morphologies observed for glucagon fibrils formed at pH 2–3. The scale bar is 50 nm. For the parallel bundled fibrils observed in a single batch, the apparent protofilament widths range from 5.2 to 6.1 nm.

Summary on how selective environmental conditions affect the preferred secondary structure of glucagon fibrils. Based on kinetics data obtained from urea and proteolysis dissociation of fibrils (), we conclude that even the most stable glucagon fibril is in equilibrium with monomers. Therefore, transition from one type of fibril to another proceeds via recycling of monomers as suggested for the SH3 domain ().

Overview of the spectroscopic properties of different glucagon fibrils. (A) CD spectra of sonicated fibrils compared to monomeric glucagon, showing the characteristic positive peak at 205 nm for type B () and the α-helix-like spectrum of sulfate-type fibrils (). The unusual spectra may be caused by overrepresentation of aromatic residues in glucagon. (B) Comparison of FTIR spectra of dried samples of different glucagon fibrils, showing both the actual spectra and deconvoluted peaks. Data taken from ,). (C) Comparison of melting curves for sonicated fibrils of types A, B, C, and D. Data adapted from .

Time course for seeded glucagon fibril formation. One gram per liter of glucagon in 50 mM glycine/HCl (pH 2.5) is seeded with a threefold dilution series of preformed fibrils. The mass fraction of preformed fibrils is indicated on the logarithmic Y-axis of the graph. The exponential growth phase extrapolates to (just above) the seed fraction added. Below seed fractions of 1:5 × 10⁻⁵, spontaneous nucleation accounts for more fibril formation than elongation of seeds. However, the shape of the observed fibrillation curve remains unchanged. At higher seed fractions the slope of the curve increases, which can be attributed to a shorter average length of the seeding fibrils relative to the average length of fibrils during the log phase.

Seeding of 1 g/L glucagon in 50 mM glycine/HCl with fivefold dilution series of sonicated fibrils. (A) Seeding with type B fibrils: At high seed fractions, daughter fibrils inherit the low ThT staining of the parent. However, at lower seed fractions, there is a transient formation of the highly ThT-fluorescent type A fibril. (B) Seeding with type D fibrils: Curve shape is dramatically different from that obtained with type B seeds. Transient formation of type A fibrils is only observed at the lowest seeding fractions. (C) Analysis of kinetics indicates that type B and D fibrils have a growth rate of 0.33 and 0.48 h⁻¹, respectively, based on the time it takes for fibrils to grow to a fixed threshold. Data taken from .