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Article: On-line coupling of continuous-flow gel electrophoresis with inductively coupled plasma-mass spectrometry to quantitatively evaluate intracellular metal binding properties of metallochaperones HpHypA and HpHspA in E. coli cells

TitleOn-line coupling of continuous-flow gel electrophoresis with inductively coupled plasma-mass spectrometry to quantitatively evaluate intracellular metal binding properties of metallochaperones HpHypA and HpHspA in E. coli cells
Authors
Issue Date2015
PublisherRoyal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/Publishing/Journals/MT/About.asp
Citation
Metallomics, 2015, v. 7 n. 10, p. 1399-1406 How to Cite?
AbstractOn-line coupling of gel electrophoresis with inductively coupled plasma-mass spectrometry (GE-ICP-MS) offers a strategy to monitor intracellular metals and their associated proteins simultaneously. Herein, we examine the feasibility of the GE-ICP-MS system in quantitative analysis of intracellular metal binding properties using two Helicobacter pylori metallochaperones HypA and HspA overexpressed in E. coli cells as a showcase. We show that parallel detection of metal and sulfur signals allows accurate quantification of intracellular metal-protein stoichiometries, even for metalloproteins that bind metal ions with micromolar affinities. Using this approach, we demonstrate that only trace amounts of Ni2+ associated with HpHypA in cells, distinct from in vitro observation of stoichiometric binding, while HpHypA exhibits a high fidelity towards its structural metal Zn2+ with stoichiometric Zn2+ binding. In contrast, HpHspA associates with Zn2+, Ni2+, Cu2+ and Co2+ from an essential metal pool with ca. 0.5 molar equivalents of total metals bound per HpHspA monomer. The metal binding properties of both HpHypA and HpHspA were altered by Bi3+. Bindings of both Zn2+ and Ni2+ to HpHypA were suppressed under the stress of Bi3+ in cells, different from in vitro studies that Bi3+ only interfered with Zn2+ but not Ni2+ binding. This study provides an analytical approach to investigate intracellular metal selectivity of overexpressed metalloproteins.
Persistent Identifierhttp://hdl.handle.net/10722/215152

 

DC FieldValueLanguage
dc.contributor.authorWANG, Y-
dc.contributor.authorHu, L-
dc.contributor.authorYANG, X-
dc.contributor.authorCHANG, YY-
dc.contributor.authorHU, X-
dc.contributor.authorLi, H-
dc.contributor.authorSun, H-
dc.date.accessioned2015-08-21T13:16:12Z-
dc.date.available2015-08-21T13:16:12Z-
dc.date.issued2015-
dc.identifier.citationMetallomics, 2015, v. 7 n. 10, p. 1399-1406-
dc.identifier.urihttp://hdl.handle.net/10722/215152-
dc.description.abstractOn-line coupling of gel electrophoresis with inductively coupled plasma-mass spectrometry (GE-ICP-MS) offers a strategy to monitor intracellular metals and their associated proteins simultaneously. Herein, we examine the feasibility of the GE-ICP-MS system in quantitative analysis of intracellular metal binding properties using two Helicobacter pylori metallochaperones HypA and HspA overexpressed in E. coli cells as a showcase. We show that parallel detection of metal and sulfur signals allows accurate quantification of intracellular metal-protein stoichiometries, even for metalloproteins that bind metal ions with micromolar affinities. Using this approach, we demonstrate that only trace amounts of Ni2+ associated with HpHypA in cells, distinct from in vitro observation of stoichiometric binding, while HpHypA exhibits a high fidelity towards its structural metal Zn2+ with stoichiometric Zn2+ binding. In contrast, HpHspA associates with Zn2+, Ni2+, Cu2+ and Co2+ from an essential metal pool with ca. 0.5 molar equivalents of total metals bound per HpHspA monomer. The metal binding properties of both HpHypA and HpHspA were altered by Bi3+. Bindings of both Zn2+ and Ni2+ to HpHypA were suppressed under the stress of Bi3+ in cells, different from in vitro studies that Bi3+ only interfered with Zn2+ but not Ni2+ binding. This study provides an analytical approach to investigate intracellular metal selectivity of overexpressed metalloproteins.-
dc.languageeng-
dc.publisherRoyal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/Publishing/Journals/MT/About.asp-
dc.relation.ispartofMetallomics-
dc.titleOn-line coupling of continuous-flow gel electrophoresis with inductively coupled plasma-mass spectrometry to quantitatively evaluate intracellular metal binding properties of metallochaperones HpHypA and HpHspA in E. coli cells-
dc.typeArticle-
dc.identifier.emailLi, H: hylichem@hku.hk-
dc.identifier.emailSun, H: hsun@hku.hk-
dc.identifier.authoritySun, H=rp00777-
dc.identifier.doi10.1039/C5MT00054H-
dc.identifier.hkuros247669-

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