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- Publisher Website: 10.1039/C5MT00054H
- Scopus: eid_2-s2.0-84944047144
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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
Title | 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 |
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Authors | |
Issue Date | 2015 |
Publisher | Royal 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? |
Abstract | On-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 Identifier | http://hdl.handle.net/10722/215152 |
ISSN | 2023 Impact Factor: 2.9 2023 SCImago Journal Rankings: 0.706 |
ISI Accession Number ID |
DC Field | Value | Language |
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dc.contributor.author | WANG, Y | - |
dc.contributor.author | Hu, L | - |
dc.contributor.author | YANG, X | - |
dc.contributor.author | CHANG, YY | - |
dc.contributor.author | HU, X | - |
dc.contributor.author | Li, H | - |
dc.contributor.author | Sun, H | - |
dc.date.accessioned | 2015-08-21T13:16:12Z | - |
dc.date.available | 2015-08-21T13:16:12Z | - |
dc.date.issued | 2015 | - |
dc.identifier.citation | Metallomics, 2015, v. 7 n. 10, p. 1399-1406 | - |
dc.identifier.issn | 1756-5901 | - |
dc.identifier.uri | http://hdl.handle.net/10722/215152 | - |
dc.description.abstract | On-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.language | eng | - |
dc.publisher | Royal Society of Chemistry. The Journal's web site is located at http://www.rsc.org/Publishing/Journals/MT/About.asp | - |
dc.relation.ispartof | Metallomics | - |
dc.title | 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 | - |
dc.type | Article | - |
dc.identifier.email | Li, H: hylichem@hku.hk | - |
dc.identifier.email | Sun, H: hsun@hku.hk | - |
dc.identifier.authority | Sun, H=rp00777 | - |
dc.identifier.doi | 10.1039/C5MT00054H | - |
dc.identifier.scopus | eid_2-s2.0-84944047144 | - |
dc.identifier.hkuros | 247669 | - |
dc.identifier.eissn | 1756-591X | - |
dc.identifier.isi | WOS:000362681200003 | - |
dc.identifier.issnl | 1756-5901 | - |