Application of Fluorescence Resonance Energy Transfer to Examine EnvZ/OmpR Interactions

S. Thomas King, Linda Kenney

Research output: Chapter in Book/Report/Conference proceedingChapter

14 Scopus citations

Abstract

The EnvZ/OmpR two-component regulatory system is best known for regulating the porin genes ompF and ompC in response to changes in the osmolarity of the growth medium. In response to an unknown signal, EnvZ is autophosphorylated by ATP on a histidine residue. The phosphoryl group is subsequently transferred to a conserved aspartate residue on OmpR. Phosphorylation of OmpR increases its affinity for the regulatory regions of the porin genes, altering their expression. Phosphorylation also alters the interaction with EnvZ and OmpR. In order to study the interactions of EnvZ and OmpR, we employed a full-length EnvZ construct fused to the green fluorescent protein (GFP) that was overexpressed and targeted to the inner membrane. Spheroplasts were prepared and lysed in microtiter plates containing purified, fluorescent-labeled OmpR protein. Fluorescence resonance energy transfer (FRET) from the GFP donor to fluorescein- or rhodamine-conjugated OmpR acceptor occurred, indicating that the two proteins interact. We then used FRET to further characterize the effect of phosphorylation on the interaction parameters. Results indicate that the full-length EnvZ behaves similarly to the isolated cytoplasmic domain EnvZc alone. Furthermore, the phospho-OmpR protein has a reduced affinity for the EnvZ kinase. This chapter describes general considerations regarding such experiments and provides detailed protocols for quantitatively measuring them.

Original languageEnglish (US)
Title of host publicationTwo Component Signaling Systems, Part A
PublisherAcademic Press Inc.
Pages352-360
Number of pages9
DOIs
StatePublished - Jan 1 2007
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume422
ISSN (Print)0076-6879

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

King, S. T., & Kenney, L. (2007). Application of Fluorescence Resonance Energy Transfer to Examine EnvZ/OmpR Interactions. In Two Component Signaling Systems, Part A (pp. 352-360). (Methods in Enzymology; Vol. 422). Academic Press Inc.. https://doi.org/10.1016/S0076-6879(06)22017-2