MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Mapping out the multistage fibrillation of glucagon. Mapping out the multistage fibrillation of glucagon. FEBS J. (1742-464X) 2012 imex curation Ghodke S., Nielsen SB., Christiansen G., Hjuler HA., Flink J., Otzen D. Only protein-protein interactions in vitro In vitro tem transmission electron microscopy tem predetermined predetermined participant predetermined Ghodke S., Nielsen SB., Christiansen G., Hjuler HA., Flink J., Otzen D. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation Mapping out the multistage fibrillation of glucagon. Mapping out the multistage fibrillation of glucagon. FEBS J. (1742-464X) 2012 imex curation Ghodke S., Nielsen SB., Christiansen G., Hjuler HA., Flink J., Otzen D. Only protein-protein interactions in vitro In vitro fluorescence fluorescence technology fluorescence predetermined predetermined participant predetermined Ghodke S., Nielsen SB., Christiansen G., Hjuler HA., Flink J., Otzen D. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation Mapping out the multistage fibrillation of glucagon. Mapping out the multistage fibrillation of glucagon. FEBS J. (1742-464X) 2012 imex curation Ghodke S., Nielsen SB., Christiansen G., Hjuler HA., Flink J., Otzen D. Only protein-protein interactions in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Ghodke S., Nielsen SB., Christiansen G., Hjuler HA., Flink J., Otzen D. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation gluc_human Pro-glucagon GCG protein protein human Homo sapiens Human MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK DIP protein P01275 original sequence version: 152 DIP protein P01275 original sequence version: 107 mint homomint 7A99EEC629B2862C gcg-gcg 3 Glucagon 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Glucagon 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain direct interaction direct interaction homomint mint We chose to study glucagon fibrillation at 3 mg/ml, wherein the soluble glucagon is largely trimeric and undergoes fibrillation at an experimentally reasonable rate so as to be able to follow the structural transitions of the peptide using several biophysical techniques. Glucagon fibrillation was monitored by circular dichroism over time. Fig. 1a shows selected representative CD spectra from a total of 23 spectra (shown in Fig. S1) following the structural stages during fibrillation. Detailed analyses of the far-UV spectra indicate numerous different structural species of glucagon through the course of fibrillation. In addition, this data is largely reproducible as exemplified by another time-series CD dataset shown in the supplementary (Fig. S4). f1 f2a t1 sf1 sf2 sf3 sf4 st1 st2 gcg-gcg-1 2 Glucagon 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Glucagon 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain direct interaction direct interaction homomint mint To complement our CD analyses, we monitored ThT, ANS and endogenous Trp fluorescence in parallel. ThT exhibits increased fluorescence on binding to fibrils [9], while ANS preferentially binds to exposed hydrophobic surfaces. Both dyes undergo a characteristic blue shift in intensity to 482 nm (ThT) and 474 nm (ANS) upon binding to glucagon aggregates at an early stage in the aggregation reaction; in the subsequent process, they undergo similar changes in intensity while retaining the same λmax. The time profiles involve an apparent lowintensity lag phase, followed by a steep rise in ANS and ThT intensity until 360 min (Fig. 2a). The increase in ANS and ThT suggest development of fibrillar structures (ThT binding) with exposed hydrophobic patches (ANS binding), closely following the β-sheet formation as shown by CD (Fig. 1c and 2a). Coinciding with decrease in β-sheet content in CD measurements (Fig. 1c), a drop in the ANS and ThT intensity is observed from 390 min until ~600 min, after which both ANS and ThT intensities stabilize at ~30-40% of the maximum intensity (Fig. 2a). This indicates structural rearrangements, eg. greater levels of intermolecular contacts, leading to fewer ThT binding sites and decreased exposure of hydrophobic patches (ANS). f2 t1 sf6 st2 gcg-gcg-2 1 Glucagon 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain Glucagon 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain direct interaction direct interaction homomint mint the TEM imaging of the fibrillating glucagon sample highlights at least three different oligomeric species and fibril morphologies with different twisting patterns. Fig. S5 describes the surface profiles of the three different oligomeric species existing in the fibrillating solution, as extracted from the TEM images, while Table 1 and S2 describes all the different molecular species observed. f4 t1 sf5 st2