UniProt UniProt http://www.uniprot.org atanassova-2005-1 in vitro In vitro chromatography chromatography technology column chromatography chromatography predetermined predetermined participant predetermined Atanassova A., Zamble DB. Protein purified by Nickel-chromatography then presence of metal assayed by either Inductively coupled plasma atomic emission spectroscopy or HPLC dzamble@chem.utoronto.ca Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation atanassova-2005-4 in vitro In vitro crosslink cross-linking study crosslink experimental particp experimental participant identification experimental particp Atanassova A., Zamble DB. Crosslinking agent - 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Stoichiometry was estimated from molecular weight of the protein(s) involved. Details of HypB construct taken from PMID:16142921 dzamble@chem.utoronto.ca Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation atanassova-2005-2 in vitro In vitro molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column predetermined predetermined participant predetermined Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation atanassova-2005-3 in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation zinc(2+) zinc(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hypa_ecoli Hydrogenase maturation factor HypA hypA b2726 JW2696 protein protein ecoli Escherichia coli (strain K12) MHEITLCQRALELIEQQAAKHGAKRVTGVWLKIGAFSCVETSSLAFCFDLVCRGSVAEGCKLHLEEQEAECWCETCQQYVTLLTQRVRRCPQCHGDMLQIVADDGLQIRRIEIDQE cobalt(2+) cobalt(2+) Co(II) Co(2+) Co2+ COBALT (II) ION Cobalt(2+) cobalt(2+) ion cobalt(II) cation cobaltous ion cobalt(II) cation cobalt(2+) ion cobalt(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hypb_ecoli Hydrogenase maturation factor HypB hypB Hydrogenase isoenzymes nickel incorporation protein HypB JW2697 b2727 protein protein ecoli Escherichia coli (strain K12) MCTTCGCGEGNLYIEGDEHNPHSAFRSAPFAPAARPKMKITGIKAPEFTPSQTEEGDLHYGHGEAGTHAPGMSQRRMLEVEIDVLDKNNRLAERNRARFAARKQLVLNLVSSPGSGKTTLLTETLMRLKDSVPCAVIEGDQQTVNDAARIRATGTPAIQVNTGKGCHLDAQMIADAAPRLPLDDNGILFIENVGNLVCPASFDLGEKHKVAVLSVTEGEDKPLKYPHMFAAASLMLLNKVDLLPYLNFDVEKCIACAREVNPEIEIILISATSGEGMDQWLNWLETQRCA nickel(2+) Nickel(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hypa-zinc(2+)-1 3637529 N/A 3637533 unspecified role unspecified role neutral component neutral component Stoichiometry: 2.0 N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Table 1, Fig. 4 Although the amount of zinc detected in HypA suggested that there were two zinc ions bound per dimer, 2 full equivalents were not detected immediately following purification. This ratio, however, was confirmed by incubation with several equivalents of zinc. Interaction observed by titrating with the metallochromic indicator PAR. Trace levels of Nickel were also observed. hypa-1 3637529 N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 5.0 direct interaction direct interaction false false false Table 1 Only low levels of zinc were observed. cobalt(2+)-hypa-1 3637529 N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 N/A 3637535 unspecified role unspecified role neutral component neutral component Stoichiometry: 1.0 direct interaction direct interaction false false false Table 1, Fig. 5 A difference spectrum of cobalt-substituted HypA was generated by subtracting the spectrum of the zinc-containing HypA from the cobalt-loaded protein hypa-hypb-2 3637530 N/A 3637536 weight by comassie molecular weight estimation by coomasie staining weight by comassie western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 N/A 3637534 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 1.0 direct interaction direct interaction false false false Fig. 8 hypb-1 3637530 N/A 3637536 weight by comassie molecular weight estimation by coomasie staining weight by comassie western blot western blot Immuno blot Immuno blot unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig. 8 hypa-10 3637530 N/A 3637534 weight by comassie molecular weight estimation by coomasie staining weight by comassie unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig. 8 hypa-2 3637531 N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 5.0 direct interaction direct interaction false false false Fig. 3 Protein purified in the presence of EDTA hypa-zinc(2+)-2 3637531 N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) Stoichiometry: 2.0 direct interaction direct interaction false false false Fig. 3 Protein purified in the presence of zinc hypa-zinc(2+)-3 3637532 N/A 3637533 unspecified role unspecified role neutral component neutral component N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 3637532 Fig. 6 Kd was measured by titrating with the metallochromic indicator PAR. hypa-nickel(2+)-1 3637532 N/A 3637537 unspecified role unspecified role neutral component neutral component N/A 3637534 unspecified role unspecified role neutral component neutral component purified purified 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 false ecoli Escherichia coli (strain K12) direct interaction direct interaction false false false 3637532 Fig. 7 Kd was measured by titrating with the metallochromic indicator PAR.