title: Structural transitions and interactions in the early stages of human glucagon amyloid fibrillation.
abstract: A mechanistic understanding of the intermolecular interactions and structural changes during fibrillation is crucial for the design of safe and efficacious glucagon formulations. Amide hydrogen/deuterium exchange with mass spectrometric analysis was used to identify the interactions and amino acids involved in the initial stages of glucagon fibril formation at acidic pH. Kinetic measurements from intrinsic and thioflavin T fluorescence showed sigmoidal behavior. Secondary structural measurement of fibrillating glucagon using far-UV circular dichroism spectroscopy showed changes in structure from random coil → α-helix → β-sheet, with increase in α-helix content during the lag phase followed by increase in β-sheet content during the growth phase. Hydrogen/deuterium exchange with mass spectrometric analysis of fibrillating glucagon suggested that C-terminal residues 22-29 are involved in interactions during the lag phase, during which N-terminal residues 1-6 showed no changes. Molecular dynamics simulations of glucagon fragments showed C-terminal to C-terminal interactions with greater α-helix content for the 20-29 fragment, with hydrophobic and aromatic residues (Phe-22, Trp-25, Val-23, and Met-27) predominantly involved. Overall, the study shows that glucagon interactions during the early phase of fibrillation are mediated through C-terminal residues, which facilitate the formation of α-helix-rich oligomers, which further undergo structural rearrangement and elongation to form β-sheet-rich mature fibrils.
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana.
authors.forename: Balakrishnan S
authors.initials: BS
authors.lastname: Moorthy
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
authors.forename: Hamed Tabatabaei
authors.initials: HT
authors.lastname: Ghomi
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
authors.forename: Markus A
authors.initials: MA
authors.lastname: Lill
authors.affiliation: Department of Industrial and Physical Pharmacy, Purdue University, West Lafayette, Indiana. Electronic address: topp@purdue.edu.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Topp
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism; D005934:Glucagon / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data
chemical_list: D000682:Amyloid; D005934:Glucagon