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

Department of Microbiology and Immunology, University of Immunology, University of Illinois-Chicago, Chicago, Illinois, USA.
Methods in Enzymology (Impact Factor: 2.09). 02/2007; 422:352-60. DOI: 10.1016/S0076-6879(06)22017-2
Source: PubMed


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.

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    • "Our HDXMS results demonstrated that conformational changes in the ATP-binding domain were independent and uncoupled from changes in the OmpR-binding site (residues 267–278; AESINKDIEECN), making it unlikely that the phosphatase activity could be regulated by osmolality and stimulated by nucleotides (Figure 5B; Supplementary Table SI). Instead, our results are consistent with an alternative view that the in-vivo level of EnvZ is too low for the phosphatase activity to be significant in signalling (Mattison and Kenney, 2002; Kenney, 2010) and that the affinity of EnvZ for OmpRBP is lower than its affinity for OmpR, which would drive the reaction in the wrong direction (Mattison and Kenney, 2002; King and Kenney, 2007). Furthermore, the results in Figure 4 demonstrate a direct effect of osmolytes on EnvZ c autophosphorylation , identifying it as the osmo-stimulated activity. "
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    ABSTRACT: Two-component systems mediate bacterial signal transduction, employing a membrane sensor kinase and a cytoplasmic response regulator (RR). Environmental sensing is typically coupled to gene regulation. Understanding how input stimuli activate kinase autophosphorylation remains obscure. The EnvZ/OmpR system regulates expression of outer membrane proteins in response to osmotic stress. To identify EnvZ conformational changes associated with osmosensing, we used HDXMS to probe the effects of osmolytes (NaCl, sucrose) on the cytoplasmic domain of EnvZ (EnvZ(c)). Increasing osmolality decreased deuterium exchange localized to the four-helix bundle containing the autophosphorylation site (His(243)). EnvZ(c) exists as an ensemble of multiple conformations and osmolytes favoured increased helicity. High osmolality increased autophosphorylation of His(243), suggesting that these two events are linked. In-vivo analysis showed that the cytoplasmic domain of EnvZ was sufficient for osmosensing, transmembrane domains were not required. Our results challenge existing claims of robustness in EnvZ/OmpR and support a model where osmolytes promote intrahelical H-bonding enhancing helix stabilization, increasing autophosphorylation and downstream signalling. The model provides a conserved mechanism for signalling proteins that respond to diverse physical and mechanical stimuli.
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