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 Synuclein as an intrinsically disordered monomer: fact or artifact? Synuclein as an intrinsically disordered monomer: fact or artifact? FEBS J. (1742-464X) 2013 imex curation Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br Accepted 2013-AUG-04 by MARTA 28-Oct_2013: Contacted by IntAct-Help. FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation Synuclein as an intrinsically disordered monomer: fact or artifact? Synuclein as an intrinsically disordered monomer: fact or artifact? FEBS J. (1742-464X) 2013 imex curation Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro ms interact detect detection by mass spectrometry ms interact detect predetermined predetermined participant predetermined Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br Accepted 2013-AUG-04 by MARTA 28-Oct_2013: Contacted by IntAct-Help. FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation Synuclein as an intrinsically disordered monomer: fact or artifact? Synuclein as an intrinsically disordered monomer: fact or artifact? FEBS J. (1742-464X) 2013 imex curation Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br Accepted 2013-AUG-04 by MARTA 28-Oct_2013: Contacted by IntAct-Help. FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation Synuclein as an intrinsically disordered monomer: fact or artifact? Synuclein as an intrinsically disordered monomer: fact or artifact? FEBS J. (1742-464X) 2013 imex curation Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br 28-Oct_2013: Contacted by IntAct-Help. Only protein-protein interactions in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Coelho-Cerqueira E., Carmo-Gonçalves P., Sá Pinheiro A., Cortines J., Follmer C. follmer@iq.ufrj.br Accepted 2013-AUG-04 by MARTA 28-Oct_2013: Contacted by IntAct-Help. FEBS J. (1742-464X) 2013 Only protein-protein interactions imex curation syua_human Alpha-synuclein Non-A beta component of AD amyloid Non-A4 component of amyloid precursor SNCA NACP PARK1 protein protein human Homo sapiens Human MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA DIP protein P37840 original sequence version: 124 DIP protein P37840 original sequence version: 175 DIP protein P37840 original sequence version: 178 6BB2F12128931663 1-134 4 aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction f1b f1c f4b Both heat- and acid-purified α-syn displayed a CD spectrum with a negative band at 200 nm that is typical of disordered structures, although a slight band at approximately 220 nm was verified for the α-syn obtained using the heating protocol (Figure 1B). Additionally, the α-syn purified by both the acidification and heating methods displayed similar fibrillogenic properties; typical sigmoidal curves for the kinetics of fibrillization were observed, with an initial lag phase, associated with the nucleation process, followed by an exponential growth phase (Figure 1C). 1-132 3 aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction f2a f2b f3g f4a To determine the oligomeric state of the recombinant α-syn purified with either the heating or the acidification protocol, the protein was cross-linked with GHD and then analyzed by SDS-PAGE (Figure 2). After cross-linking, a smallpopulation of α-syn was found as dimers (3-5% of total α-syn) with an apparent molecular mass of ~34 kDa, as determined using the Totallab software (Figures 2A and 2B). These species were also revealed by western blot using the monoclonal antibody anti-human α-syn 14H2L1. We also observed a very small population of α-syn running at ~60 kDa. Because α-syn monomers and dimers exhibit respective molecular masses of 18 and 34 kDa according to SDS-PAGE, this 60 kDa protein may be either a tetrameric or trimeric form of α-syn. 1-133 1 aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction f3d f3e f3f Collectively, these data provide convincing evidence that α-syn dimers are highly dynamic and exist in equilibrium with the monomeric species 1-131 2 aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction f2c Mass spectrometry (MALDI-TOF/TOF) was used to confirm the oligomeric state of cross-linked α-syn. A molecular mass of 14,458 Da was obtained from the MALDI-TOF/TOF spectrum of non-cross-linked α-syn purified using the heating protocol (Figure 2C), and no differences were observed in comparison with α- syn purified using the acidification protocol (data not shown). After cross-linking, both heat- and acid-purified α-syn exhibited a minor population of dimers and trimers with the predominant monomer. For the α-syn purified using the heating protocol, a very small population of tetramers (64,269 Da) was also observed. Together, the analysis of crosslinked α-syn by both SDS-PAGE and mass spectroscopy indicated that the protein is mostly monomeric with a small population of oligomers, notably dimers. 123-1 2 aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) aplha-Syn 5 unspecified role unspecified role neutral component neutral component purified purified ecoli Escherichia coli (strain K12) direct interaction direct interaction f2c Mass spectrometry (MALDI-TOF/TOF) was used to confirm the oligomeric state of cross-linked α-syn. A molecular mass of 14,458 Da was obtained from the MALDI-TOF/TOF spectrum of non-cross-linked α-syn purified using the heating protocol (Figure 2C), and no differences were observed in comparison with α- syn purified using the acidification protocol (data not shown). After cross-linking, both heat- and acid-purified α-syn exhibited a minor population of dimers and trimers with the predominant monomer. For the α-syn purified using the heating protocol, a very small population of tetramers (64,269 Da) was also observed. Together, the analysis of crosslinked α-syn by both SDS-PAGE and mass spectroscopy indicated that the protein is mostly monomeric with a small population of oligomers, notably dimers.