Glucagon fibril polymorphism reflects differences in protofilament backbone structure.
Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional beta-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from beta-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures.
Andersen
Christian Beyschau
CB
Protein Structure and Biophysics, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark.
Hicks
Matthew R
MR
Vetri
Valeria
V
Vandahl
Brian
B
Rahbek-Nielsen
Henrik
H
Thøgersen
Henning
H
Thøgersen
Ida Bukh
IB
Enghild
Jan Johannes
JJ
Serpell
Louise C
LC
Rischel
Christian
C
Otzen
Daniel Erik
DE
D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D005934:Glucagon / Q000737:chemistry* / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017550:Spectroscopy, Fourier Transform Infrared; D014961:X-Ray Diffraction
D000682:Amyloid; D005934:Glucagon; D010447:Peptide Hydrolases