31P and 13C solid-state NMR analysis of morphological changes of phospholipid bilayers containing glucagon during fibril formation of glucagon under neutral condition

Glucagon is a 29 amino acid peptide hormone secreted by pancreatic alpha-cells that interacts with specific receptors located in various organs. Glucagon tends to form gel-like fibrillar aggregates that are cytotoxic due to their activation of apoptotic signaling pathways. To understand the glucagon-membrane interactions, morphological changes in dimyristoylphosphatidylcholine (DMPC) bilayers containing glucagon in neutral solution were investigated by observing P-31 NMR spectra. First, lipid bilayers with a DMPC/glucagon molar ratio of 50/1 were observed. One day after preparing the DMPC/glucagon lipid bilayer sample, lipid bilayers were disrupted below the phase transition temperature (Tc). Membrane disruption was reduced 2 days after preparation due to the reduction of glucagon-DMPC interaction, and subsequently increased by 4 days and was reduced again by 7 days. TEM measurements showed that small ellipsoidal intermediates of glucagon were observed inside the small size of lipid bilayer after 4 days, while fibrils grew inside lipid bilayer after 19 days. These results indicate that morphological changes in DMPC/glucagon lipid bilayers are correlated with the evolution of glucagon aggregate state. Particularly, fibril intermediate shows a strong glucagon lipid bilayer interaction. We further investigated the structure and kinetics of glucagon fibril formation inside the DMPC lipid bilayer in a neutral solution using C-13 solid-state NMR spectroscopy. alpha-Helical structures were observed around Gly4 and Ala19 in the monomeric form, which changed to beta-sheet structures in the fibril form. The fibrillation process can be explained by a two-step autocatalytic reaction mechanism in which the first step is a homogeneous nuclear formation (k(1)), and the second step is an autocatalytic heterogeneous fibrillation process (k(2)).