Early Stages of Amyloid Fibril Formation Studied by Liquid-State NMR: The Peptide Hormone Glucagon

Abstract

The 29-residue peptide hormone glucagon forms amyloid fibrils within a few hours at low pH. In this study, we use glucagon as a model system to investigate fibril formation by liquid-state (1)H-NMR spectroscopy One-dimensional, correlation, and diffusion experiments monitoring the fibril formation process provide insight into the early stages of the pathway on which the molecules aggregate to fibrils. In conjunction with these techniques, exchange experiments give information about the end-state conformation. Within the limits of detection, there are no signs of larger oligomeric intermediates in the course of the fibril formation process. Kinetic information is extracted from the time course of the residual free glucagon signal decay. This suggests that glucagon amyloids form by a nucleated growth mechanism in which trimers (rather than monomers) of glucagon interact directly with the growing fibrils rather than with each other. The results of proton/deuterium exchange experiments on mature fibrils with subsequent dissolution show that the N-terminal of glucagon is the least amenable to exchange, which indicates that this part is strongly involved in the intermolecular bonds of the fibrils.

Nanomechanics of individual amyloid fibrils using atomic force microscopy

“…Using electron microscopy, atomic force microscopy (AFM), X-ray diffraction and solid state NMR, the morphology and structures of the amyloid fibers have been extensively studied and a wealth of information have been accumulated [3,4]. It is widely accepted that amyloid fibrils have a common core structure with β-strands perpendicular to the fibril axis, and the fibrils have been shown with one or more protofilaments intertwisting together [5].…”

Structural Transitions and Interactions in the Early Stages of Human Glucagon Amyloid Fibrillation

“…5 B). Peptic fragments 1-6, 1-9, and 10-21 still showed~25%,~14%, and~10% deuterium uptake, respectively, after 1440 min, whereas deuterium incorporation for the C-terminus (residues [22][23][24][25][26][27][28][29] was <1% after only 480 min of fibrillation (Figs. 5 A and S4).…”

“…Though there have been numerous studies of glucagon fibrillation (23)(24)(25)(26)(27)(28)(29), the molecular mechanisms involved have not been well studied, due in part to the rapid fibrillation kinetics and the lack of high-resolution methods. As with other amyloidogenic peptides and proteins, glucagon fibrillation is a complex process.…”

“…Complexity is further increased by structural polymorphism in the fibrils themselves, which results from changes in concentration, solvent composition, and temperature (24,30). Several studies have shown that both hydrophobic and charged amino acids in the glucagon sequence are critical for fibrillation (28,29,(31)(32)(33)(34). Some suggest that the hydrophobic residues in the N-and C-terminus are involved (32), whereas others argue that only the N-terminal region participates (28).…”

“…Several studies have shown that both hydrophobic and charged amino acids in the glucagon sequence are critical for fibrillation (28,29,(31)(32)(33)(34). Some suggest that the hydrophobic residues in the N-and C-terminus are involved (32), whereas others argue that only the N-terminal region participates (28). The results from these studies have been based either on analysis of mutants or characterization of the mature fibrils, rather than on studies of native glucagon itself, and may depend on solution composition and temperature.…”