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Volume 151, Issue 12

Dimerization and DNA binding of the PhoP response regulator are phosphorylation independent Free

Abstract

In , PhoP is the response regulator of the PhoP/PhoQ two-component regulatory system that controls the expression of various virulence factors in response to external Mg. Previous studies have shown that phosphorylation of a PhoP variant with a C-terminal His tag (PhoP) enhances dimerization and binding to target DNA. Here, the effect of phosphorylation on the oligomerization and DNA binding properties of both wild-type PhoP (PhoP) and PhoP are compared. Gel filtration chromatography showed that PhoP exists as a mixture of monomer and dimer regardless of its phosphorylation state. In contrast, unphosphorylated PhoP was mostly monomeric, whereas PhoP∼P existed as a mixture of monomer and dimer. By monitoring the tryptophan fluorescence of the proteins and the fluorescence of the probe 1-anilinonaphthalene-8-sulfonic acid bound to them, it was found that PhoP and PhoP exhibited different spectral properties. The interaction between PhoP or PhoP and the PhoP box of the promoter was monitored by surface plasmon resonance. Binding of PhoP to the PhoP box was barely influenced by phosphorylation. In contrast, phosphorylation of PhoP clearly increased the interaction of PhoP with target DNA. Altogether, these data show that a His tag at the C-terminus of PhoP affects its biochemical properties, most likely by affecting its conformation and/or its oligomerization state. More importantly, these results show that wild-type PhoP dimerization and interaction with target DNA are independent of phosphorylation, which is in contrast to the previously proposed model.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
Keyword(s): ANS, 1-anilinonaphthalene-8-sulfonic acid , GST, glutathione S-transferase , RU, resonance unit and SPR, surface plasmon resonance
SGM
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2005-12-01
2024-04-27
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Dimerization and DNA binding of the Salmonella enterica PhoP response regulator are phosphorylation independent
Microbiology 151, 3979 (2005); https://doi.org/10.1099/mic.0.28236-0
/content/journal/micro/10.1099/mic.0.28236-0
/content/journal/micro/10.1099/mic.0.28236-0
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