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.

The peptide hormone glucagon forms amyloid fibrils with two coexisting β-strand conformations

“…At moderately higher concentrations glucagon forms α-helical trimers 46 (Fig. 4f), which are likely the species responsible for initiating fibril growth 47,48 . What is the cause of glucagon’s ability to convert among these multiple conformations, especially into a stable fibril with an unprecedented β-strand length?…”

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

“…The following are the steps in the analysis: generation of a peptide list, identification of target peptides in the LC-MS data, extraction of features (isotopic patterns), calculation of deuteration levels, and representation of the raw data. Sometimes kinetic parameters for the H/D exchange process itself and the conformational changes of the protein can also be deduced from HDX-MS data [ 1 , 9 , 23 , 25 , 40 , 47 , 58 , 83 , 84 , 85 , 86 ]. For peptide identification, a theoretical and an experimental peptide list can be used together.…”

“…These types of changes are quite cumbersome to study via crystallographic techniques, but are readily assessable with HDX-MS. One of the classic uses of HDX-MS is in fact to compare conformers of proteins. This application is used in, e.g., analysis of protein dynamics [ 25 , 54 , 116 ], investigation of single nucleotide polymorphisms and their effects in disease states [ 59 , 68 , 117 ], and exploration of the mechanisms of protein aggregation and amyloid fiber formation [ 84 , 86 , 118 ]. HDX-MS is used in conjunction with native mass spectrometry and chemical cross-linking to better understand the changes in a protein structure [ 119 , 120 ].…”

Stability of Commercially Available Glucagon Formulation for Dual-Hormone Artificial Pancreas Clinical Use

“…Sterility testing according to United States Pharmacopeia USP <71> guidelines 21 : a growth promotion test was first conducted to validate the inability of glucagon to inhibit the growth of microorganisms. Briefly, reconstituted glucagon vials (1 mL) were filtered under sterile conditions and incubated separately with <100 colony-forming units of six different microorganisms in their corresponding culture medium as detailed in Supplementary Table S2 (Supplementary Data are available online at www.liebertpub.com/dia).…”

“…The fluorescence spectrum of the Trp-25 residue typically peaks at a maximum of around 350 nm and is blue shifted to *320 nm when incorporated into a fibril structure. 21 Samples of 100 lL were loaded in triplicates in 96well plates and read by Perkin Elmer Envision 2104 multilabel reader spectrophotometer system (Perkinelmer, Inc., Waltham, MA) with excitation at 280 nm and emission at a spectrum ranging from 300 to 450 nm (4 nm increments), and peaks of maximum fluorescence were recorded for each sample.…”