Surface plasmon resonance and isothermal titration calorimetry to monitor the Ni(II)-dependent binding of Helicobacter pylori NikR to DNA

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• 作者：Edoardo Fabini ; Barbara Zambelli ; Luca Mazzei…
• 关键词：Molecular recognition process ; Protein ; DNA interaction ; Binding affinity ; Helicobacter pylori NikR ; SPR ; ITC
• 刊名：Analytical and Bioanalytical Chemistry
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：408
• 期：28
• 页码：7971-7980
• 全文大小：1,497 KB
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  Food Science
  Inorganic Chemistry
  Physical Chemistry
  Monitoring, Environmental Analysis and Environmental Ecotoxicology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1618-2650
• 卷排序：408

文摘

NikR is a transcription factor that regulates the expression of Ni(II)-dependent enzymes and other proteins involved in nickel trafficking. In the human pathogenic bacterium Helicobacter pylori, NikR (HpNikR) controls, among others, the expression of the Ni(II) enzyme urease by binding the double-strand DNA (dsDNA) operator region of the urease promoter (OP_ureA) in a Ni(II)-dependent mode. This article describes the complementary use of surface plasmon resonance (SPR) spectroscopy and isothermal titration calorimetry (ITC) to carry out a mechanistic characterization of the HpNikR–OP_ureA interaction. An active surface was prepared by affinity capture of OP_ureA and validated for the recognition process in the SPR experiments. Subsequently, the Ni(II)-dependent affinity of the transcription factor for its operator region was assessed through kinetic evaluation of the binding process at variable Ni(II) concentrations. The kinetic data are consistent with a two-step binding mode involving an initial encounter between the two interactants, followed by a conformational rearrangement of the HpNikR–OP_ureA complex, leading to high affinity binding. This conformational change is only observed in the presence of the full set of four Ni(II) ions bound to the protein. The SPR assay developed and validated in this study constitutes a suitable method to screen potential drug lead candidates acting as inhibitors of this protein–dsDNA interaction.