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

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.