N- and C-terminal hydrophobic patches are involved in fibrillation of glucagon.
Recent work suggests that the molecular structure of amyloid-like fibrils is determined by environmental conditions as well as amino acid sequence. To probe the involvement of side chains in fibrillation of the 29-residue hormone glucagon, we have measured fibrillation kinetics of 15 alanine mutants. At acidic pH, all of the mutants are able to form fibrils. However, substitution of hydrophobic residues in the N- and C-termini (in particular Phe6, Tyr10, Val23, and Met27) decelerates fibrillation dramatically. This indicates that the hydrophobicity and/or high beta-sheet propensity of these residues may be important for fibrillation. In contrast, substitution of Leu14 increases fibrillation propensity compared to that of the wild type. Nevertheless, despite identical fibrillation conditions, the thioflavin T and tryptophan fluorescence spectra of fibrils formed by mutants Tyr13, Leu14, and Asp15 are significantly different from those of other mutants, indicating that substitution of these residues may influence not only the fibrillation kinetics and fibril stability but also the preferred final structure of the fibrils that is formed, in line with the general structural polymorphism of glucagon fibrils. In contrast, under alkaline conditions, only a handful of the alanine mutants are capable of forming fibrils, suggesting that more side chains are involved in stabilizing interactions here. In addition, fibrils formed by wild-type glucagon at alkaline pH appear very stable, compared to fibrils formed at acidic pH. This suggests that the distribution of charges determines the number of different fibrillated states available to a peptide, since these can block formation of metastable fibrillated states.
Pedersen
Jesper Søndergaard
JS
Center for Insoluble Protein Structures (inSPIN), Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Dikov
Dancho
D
Otzen
Daniel Erik
DE
D000143:Acids; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000468:Alkalies; D052160:Benzothiazoles; D005934:Glucagon / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013844:Thiazoles
D000143:Acids; D000468:Alkalies; D052160:Benzothiazoles; D013844:Thiazoles; C009462:thioflavin T; D005934:Glucagon; D000409:Alanine