The changing face of glucagon fibrillation: structural polymorphism and conformational imprinting.
We have established a time-resolved fluorescence assay to study fibrillation of the 29 residue peptide hormone glucagon under a variety of different conditions in a high-throughput format. Fibrils formed at pH 2.5 differ in fibrillation kinetics, morphology, thioflavin T staining and FTIR/CD spectra depending on salts, glucagon concentration and fibrillation temperature. Apparent fibrillar stability correlates with spectral and kinetic properties; generally, fibrils formed under conditions favourable for rapid fibrillation (ambient temperatures, high glucagon concentration or high salt concentration) appear less thermostable than those formed under more challenging conditions (high temperatures, low glucagon or low salt concentrations). Properties of preformed fibrils used for seeding are inherited in a prion-like manner. Thus, we conclude that the structure of fibrils formed by glucagon is not the result of the global energy minimization, but rather kinetically controlled by solvent conditions and seed-imprinting. Fibrillar polymorphism, which is being reported for an increasing number of proteins, probably reflects that fibrils have not been under evolutionary constraints to retain a single active conformation. Our results highlight the complexity of the fibrillation mechanism of glucagon, since even subtle changes in fibrillation conditions can alter the type of fibrils formed, or result in formation of mixtures of several types of fibrils.
Pedersen
Jesper Søndergaard
JS
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Dikov
Dantcho
D
Flink
James L
JL
Hjuler
Hans Aage
HA
Christiansen
Gunna
G
Otzen
Daniel Erik
DE
D000595:Amino Acid Sequence; D001704:Biopolymers / Q000737:chemistry; D002942:Circular Dichroism; D019281:Dimerization; D005934:Glucagon / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012965:Sodium Chloride / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D014508:Urea / Q000737:chemistry
D001704:Biopolymers; D012965:Sodium Chloride; D014508:Urea; D005934:Glucagon