IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. J. Biol. Chem. (0021-9258) 2002 imex curation Berkowitz O., Wirtz M., Wolf A., Kuhlmann J., Hell R. hell@ipk-gatersleben.de orchard 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 Berkowitz O., Wirtz M., Wolf A., Kuhlmann J., Hell R. hell@ipk-gatersleben.de Accepted 2009-AUG-10 by ORCHARD. J. Biol. Chem. (0021-9258) 2002 orchard Only protein-protein interactions imex curation Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana. J. Biol. Chem. (0021-9258) 2002 imex curation Berkowitz O., Wirtz M., Wolf A., Kuhlmann J., Hell R. hell@ipk-gatersleben.de orchard Only protein-protein interactions ecoli Escherichia coli (strain K12) pull down pull down weight by comassie molecular weight estimation by coomasie staining weight by comassie Berkowitz O., Wirtz M., Wolf A., Kuhlmann J., Hell R. hell@ipk-gatersleben.de Accepted 2009-AUG-10 by ORCHARD. J. Biol. Chem. (0021-9258) 2002 orchard Only protein-protein interactions imex curation sat3_arath Serine acetyltransferase 3, mitochondrial SAT3 SAT1 SATA MJG19.6 At3g13110 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MLPVTSRRHFTMSLYMLRSSSPHINHHSFLLPSFVSSKFKHHTLSPPPSPPPPPPMAACIDTCRTGKPQISPRDSSKHHDDESGFRYMNYFRYPDRSSFNGTQTKTLHTRPLLEDLDRDAEVDDVWAKIREEAKSDIAKEPIVSAYYHASIVSQRSLEAALANTLSVKLSNLNLPSNTLFDLFSGVLQGNPDIVESVKLDLLAVKERDPACISYVHCFLHFKGFLACQAHRIAHELWTQDRKILALLIQNRVSEAFAVDFHPGAKIGTGILLDHATAIVIGETAVVGNNVSILHNVTLGGTGKQCGDRHPKIGDGVLIGAGTCILGNITIGEGAKIGAGSVVLKDVPPRTTAVGNPARLLGGKDNPKTHDKIPGLTMDQTSHISEWSDYVI DIP protein Q39218 original sequence version: 81 A3ED09DEA53C10E2 cyskm_arath Cysteine synthase, mitochondrial Beta-substituted Ala synthase 2;2 OASC ACS 1 F24G16.30 At3g59760 O-acetylserine sulfhydrylase CSase C O-acetylserine (thiol)-lyase OAS-TL C protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MVAMIMASRFNREAKLASQILSTLLGNRSCYTSMAATSSSALLLNPLTSSSSSSTLRRFRCSPEISSLSFSSASDFSLAMKRQSRSFADGSERDPSVVCEAVKRETGPDGLNIADNVSQLIGKTPMVYLNSIAKGCVANIAAKLEIMEPCCSVKDRIGYSMVTDAEQKGFISPGKSVLVEPTSGNTGIGLAFIAASRGYRLILTMPASMSMERRVLLKAFGAELVLTDPAKGMTGAVQKAEEILKNTPDAYMLQQFDNPANPKIHYETTGPEIWDDTKGKVDIFVAGIGTGGTITGVGRFIKEKNPKTQVIGVEPTESDILSGGKPGPHKIQGIGAGFIPKNLDQKIMDEVIAISSEEAIETAKQLALKEGLMVGISSGAAAAAAIKVAKRPENAGKLIAVVFPSFGERYLSTPLFQSIREEVEKMQPEV DIP protein Q43725 original sequence version: 102 9DDEB1B8E314E143 sat3-oasc-1 2 4 unspecified role unspecified role prey prey 3 unspecified role unspecified role bait bait n-terminal ha tag ha tag YPYDVPDYA epitope tag YPYDVPDYA epitope tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range physical association physical association Figure 2 The authors demonstrated that OAS-TL was specifically and completely dissociated from column-bound SAT by the reaction intermediate OAS and regained full activity as a free homodimer. The mitochondrial cysteine synthase complex from A. thaliana and its single components SAT and OAS-TL can be functionally co-expressed in E. coli and purified with high purity and yield by nickel-affinity chromatograph sat3-oasc-2 1 3 unspecified role unspecified role bait bait n-terminal his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag n-term range n-terminal range n-term range n-term range n-terminal range n-term range ecoli Escherichia coli (strain K12) 4 unspecified role unspecified role prey prey ecoli Escherichia coli (strain K12) direct interaction direct interaction Figure 4 In SAT/OAS-TL association experiments the different OAS-TL concentrations applied correlated with increasing slopes of the association sensorgrams association rate constant ka = 5.1 x 10-4 M-1 S-1 The authors concluded that the linear association kinetics over a 10-fold concentration range of the analyte OAS-TL strongly suggests that a simple binding model is applicable to describe the interaction between SAT and OAS-TL oligomers as proposed by a model where the assumption of an A + B AB binding model where A would represent a SAT tetramer and B an OAS-TL dimer. They noted " Although the dissociation phase showed some deviation from the A + B AB model in very late stages (>900 s), its kinetic analysis still provided a good approximation". See text for more details. dissociation rate constant kd = 1.3 x 10-3 S-1 equilibrium dissociation constant (KD) = 25 +/- 4 x 10-9 M (n = 4).