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 PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation. PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation. FEBS J. (1742-464X) 2012 imex curation Pedersen LR., Nielsen SB., Hansted JG., Petersen TE., Otzen DE., Sørensen ES. 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 Pedersen LR., Nielsen SB., Hansted JG., Petersen TE., Otzen DE., Sørensen ES. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation. PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation. FEBS J. (1742-464X) 2012 imex curation Pedersen LR., Nielsen SB., Hansted JG., Petersen TE., Otzen DE., Sørensen ES. Only protein-protein interactions in vitro In vitro fluorescence fluorescence technology fluorescence predetermined predetermined participant predetermined Pedersen LR., Nielsen SB., Hansted JG., Petersen TE., Otzen DE., Sørensen ES. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation. PP3 forms stable tetrameric structures through hydrophobic interactions via the C-terminal amphipathic helix and undergoes reversible thermal dissociation and denaturation. FEBS J. (1742-464X) 2012 imex curation Pedersen LR., Nielsen SB., Hansted JG., Petersen TE., Otzen DE., Sørensen ES. Only protein-protein interactions in vitro In vitro cd circular dichroism CD CD cd predetermined predetermined participant predetermined Pedersen LR., Nielsen SB., Hansted JG., Petersen TE., Otzen DE., Sørensen ES. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation glcm1_bovin Glycosylation-dependent cell adhesion molecule 1 Lactophorin GLYCAM1 28 kDa milk glycoprotein PP3 Proteose-peptone component 3 protein protein bovin Bos taurus (Bovine) Bovine MKFLCVLLLASLAATSLAILNKPEDETHLEAQPTDASAQFIRNLQISNEDLSKEPSISREDLISKEQIVIRSSRQPQSQNPKLPLSILKEKHLRNATLGSEETTEHTPSDASTTEGKLMELGHKIMRNLENTVKETIKYLKSLFSHAFEVVKT mint 6679E5F1F99CB886 glycam1-glycam1 1 PP3 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified PP3 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction f3 mint Monomeric glycosylated PP3 has a molecular weight of 19.4 kDa [24], however, SEC analysis of PP3 showed a major peak eluting at 12.06 mL corresponding to a relative MW of ~191 kDa. A smaller peak eluted at 15.02 mL corresponding to ~58 kDa (Fig. 3).This indicates that PP3 preferentially assembles 11 into multimeric structures under these conditions glycam1-1 1 PP3 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 4.0 direct interaction direct interaction f4 mint Asymmetrical flow field-flow fractionation (AF4) is a size separation technique similar to size exclusion chromatography (SEC). However, unlike SEC, separation by the AF4 method has no stationary phase to influence on separation, allowing separation of sticky samples and samples sensitive to shearing forces. When coupled with RI, UV and light scattering detectors, this allows quantification and size estimates of proteins and protein complexes/aggregates from a lower limit determined by the ultracentrifugation membrane (i.e. down to ~1 kDa) and up to well above 10 MDa within the same analysis and sample [54-PMID: 15296962]. By AF4-MALS, the MW can be determined independently from light scattering data 17 by extrapolation to zero sample concentration and zero scattering angle using the 18 Zimm model. This MALS-based analysis requires the sample concentration to be 19 determined either by refractive index (RI) (based on both protein and carbohydrate 20 contributions, see Materials and Methods) or absorbance at 205 nm (giving only 21 protein contributions). In separate experiments exemplified in Fig 4B, the MW of the 22 main PP3 peak was determined in combination with RI data to 79.1±2.6 kDa (average 23 ± standard error of three individual analysis) using the weight averaged (dn/dc)PP3. 24 This value corresponds to approximately four PP3 units of ~19.8±0.7 kDa protein 25 assembled into a tetrameric complex glycam1-1 2 PP3 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 4.0 direct interaction direct interaction f5 mint As shown in Fig 19 5, the anisotropy decreases above ~1 M GdmCl, clearly indicating that PP3 tumbles 20 faster in solution under these conditions. While protein unfolding is expected to 21 increase the hydrodynamic radius of the protein and thus decrease the tumbling rate 22 (larger anisotropy), the decrease in anisotropy are consistent with dissociation of the 23 tetrameric complex. glycam1-1 3 PP3 4 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified Stoichiometry: 4.0 direct interaction direct interaction mint Consistently, unfolding transitions in GdmCl monitored by far-UV CD 5 showed a midpoint shift from 1.75±0.05 M at 3 μM to 2.16±0.04 M at 50 μM (Fig. 5) 6 (the anisotropy signal at 3 μM was too weak to allow us to follow tetramer 7 dissociation at this concentration). These sets of data show that stability increases 8 with protein concentration, i.e. unfolding is associated with dissociation. CD 9 wavescans of the PP3 over the range 2.1-30 μM showed essentially no difference in 10 the CD spectra (Fig. 6B). This observation, supported by the constant ratio between 11 monomeric and tetrameric PP3 in the AF4 chromatograms over a wide concentration 12 range (see above), reveals that the PP3 tetramer is relatively stable in the absence of 13 denaturing agents. f5 f6