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 Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein. Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein. FEBS J. (1742-464X) 2011 imex curation Leelawatwattana L., Praphanphoj V., Prapunpoj P. Only protein-protein interactions in vitro In vitro spr surface plasmon resonance Optical biosensor BIAcore(r) BIAcore(r) Optical biosensor spr predetermined predetermined participant predetermined Leelawatwattana L., Praphanphoj V., Prapunpoj P. FEBS J. (1742-464X) 2011 Only protein-protein interactions imex curation Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein. Effect of the N-terminal sequence on the binding affinity of transthyretin for human retinol-binding protein. FEBS J. (1742-464X) 2011 imex curation Leelawatwattana L., Praphanphoj V., Prapunpoj P. Only protein-protein interactions in vitro In vitro bn-page blue native page bn-page predetermined predetermined participant predetermined Leelawatwattana L., Praphanphoj V., Prapunpoj P. FEBS J. (1742-464X) 2011 Only protein-protein interactions imex curation tthy_cropo Transthyretin TTR Prealbumin protein protein cropo Crocodylus porosus MAFHSMLLVFLAGLVFLTEAAPLVSHGSIDSKCPLMVKVLDAVRGSPAANVAIKVFKKTSDGDWQEFAAGKTTEFGEVHELTSDEKFVEGIYRVEFDTSSYWKALGLSPFHEYADVVFTANDSGHRHYTIAALLSPFSYSTTAVVSDPQE mint E05DFAA6FBDE22A5 tthy_human Transthyretin Prealbumin TBPA ATTR TTR PALB protein protein human Homo sapiens Human MASHRLLLLCLAGLVFVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE DIP protein P02766 original sequence version: 114 DIP protein P02766 original sequence version: 163 DIP protein P02766 original sequence version: 174 3A6AEBCBBA56BC44 ret4_human Retinol-binding protein 4 RBP4 Plasma retinol-binding protein PRO2222 protein protein human Homo sapiens Human MKWVWALLLLAALGSGRAERDCRVSSFRVKENFDKARFSGTWYAMAKKDPEGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTEDPAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADSYSFVFSRDPNGLPPEAQKIVRQRQEELCLARQYRLIVHNGYCDGRSERNLL 660C6DD8CC9B811A ttr-1 2 TTR 3 polyclonal western polyclonal antibody western blot polyclonal western 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 Stoichiometry: 2.0 direct interaction direct interaction f1a f1b mint ttr-1 2 TTR 4 western blot western blot Immuno blot Immuno blot 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 Stoichiometry: 2.0 direct interaction direct interaction f1c mint rbp4-ttr-9 2 TTR 3 western blot western blot Immuno blot Immuno blot 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 RBP 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified direct interaction direct interaction mint f2 f3a The Scatchard plot [30] was used to determine the dissociation constant (Kd) of the binding between TTR and RBP. Moles of bound TTR were directly calculated from the fluorescence intensity of the protein band after correction for the efficiency of protein transfer. These values were assumed to be moles of bound RBP in the Scatchard equation based on 1:2 molar ratio of TTR and RBP in the complex. Free RBP was obtained by subtraction of bound RBP from the total. Non-specific binding was extrapolated and other corrections were performed prior to analysis by Scatchard plot. Least squares linear regression lines were calculated using the statistic package in Microsoft Excel and Kd values were derived from the slopes rbp4-ttr-10 2 RBP 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component purified purified TTR 4 western blot western blot Immuno blot Immuno blot 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 Scatchard plot [30] was used to determine the dissociation constant (Kd) of the binding between TTR and RBP. Moles of bound TTR were directly calculated from the fluorescence intensity of the protein band after correction for the efficiency of protein transfer. These values were assumed to be moles of bound RBP in the Scatchard equation based on 1:2 molar ratio of TTR and RBP in the complex. Free RBP was obtained by subtraction of bound RBP from the total. Non-specific binding was extrapolated and other corrections were performed prior to analysis by Scatchard plot. Least squares linear regression lines were calculated using the statistic package in Microsoft Excel and Kd values were derived from the slopes f2 f3d rbp4-ttr-11 1 TTR 3 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain RBP 5 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified direct interaction direct interaction f4 mint To confirm the changes in binding strength, the bindings of human TTR, C. porosus TTR and hu/crocTTR to human RBP were determined by SPR and compared. In agreement with the Western and ECL analysis, the sensogram showed that C. porosus TTR bound to human RBP better than human TTR did (Fig. 4), and this ability decreased when the Nterminal sequence of the reptile TTR was changed to that of human TTR. This change in amino acid sequence decreases the affinity of C. porosus TTR by a factor of ~1.4 (Kds of C. porosus TTR and hu/crocTTR were 7.31 nM and 10.54 nM, respectively). Since human TTR showed very low response even the chip was coated with 80 nM RBP, its Kd was not determined