UniProt UniProt http://www.uniprot.org Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Journal of bacteriology 2005 imex curation Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Only protein-protein interactions imex curation in vitro In vitro fluorescence spectr classical fluorescence spectroscopy fluorescence spectr predetermined predetermined participant predetermined Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Accepted 2020-MAY-20 AT 15:56 BST AT 15:56 BST by KALPANAP Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Journal of bacteriology 2005 imex curation Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Only protein-protein interactions imex curation in vitro In vitro crosslink cross-linking study crosslink weight by comassie molecular weight estimation by coomasie staining weight by comassie 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 Accepted 2020-MAY-20 AT 15:56 BST AT 15:56 BST by KALPANAP Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Journal of bacteriology 2005 imex curation Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Only protein-protein interactions imex curation 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 Accepted 2020-MAY-20 AT 15:56 BST AT 15:56 BST by KALPANAP Journal of bacteriology 2005 Only protein-protein interactions imex curation imex curation Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel. Journal of bacteriology 2005 imex curation Atanassova A., Zamble DB. dzamble@chem.utoronto.ca Only protein-protein interactions imex curation 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 Accepted 2020-MAY-20 AT 15:56 BST AT 15:56 BST by KALPANAP 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 57458941783073D4 hypb_ecoli Hydrogenase maturation factor HypB hypB Hydrogenase isoenzymes nickel incorporation protein HypB JW2697 b2727 protein protein ecoli Escherichia coli (strain K12) MCTTCGCGEGNLYIEGDEHNPHSAFRSAPFAPAARPKMKITGIKAPEFTPSQTEEGDLHYGHGEAGTHAPGMSQRRMLEVEIDVLDKNNRLAERNRARFAARKQLVLNLVSSPGSGKTTLLTETLMRLKDSVPCAVIEGDQQTVNDAARIRATGTPAIQVNTGKGCHLDAQMIADAAPRLPLDDNGILFIENVGNLVCPASFDLGEKHKVAVLSVTEGEDKPLKYPHMFAAASLMLLNKVDLLPYLNFDVEKCIACAREVNPEIEIILISATSGEGMDQWLNWLETQRCA DIP protein P0AAN3 original sequence version: 30 5B3AEB59AA6915C3 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) nickel(2+) Nickel(2+) small molecule small molecule chemical synthesis Chemical synthesis (Chemical synthesis) hypa-zinc(2+)-1 3 5 unspecified role unspecified role neutral component neutral component 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction 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 3 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Table 1 Only low levels of zinc were observed. hypa-2 4 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3 Protein purified in the presence of EDTA hypa-zinc(2+)-2 4 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 3 Protein purified in the presence of zinc cobalt(2+)-hypa-1 3 6 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 ecoli Escherichia coli (strain K12) 8 unspecified role unspecified role neutral component neutral component direct interaction direct interaction 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-zinc(2+)-3 1 5 unspecified role unspecified role neutral component neutral component 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 6 Kd was measured by titrating with the metallochromic indicator PAR. hypa-nickel(2+)-1 1 9 unspecified role unspecified role neutral component neutral component 6 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 7 Kd was measured by titrating with the metallochromic indicator PAR. hypa-hypb-2 2 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp 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 ecoli Escherichia coli (strain K12) 6 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 8 hypb-1 2 7 weight by comassie molecular weight estimation by coomasie staining weight by comassie western blot western blot Immuno blot Immuno blot experimental particp experimental participant identification experimental particp 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 8 hypa-10 2 6 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp 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 ecoli Escherichia coli (strain K12) direct interaction direct interaction Fig. 8