Application of fluorescence resonance energy transfer to examine EnvZ/OmpR interactions
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.
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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.The EMBO Journal 04/2012; 31(11):2648-59. DOI:10.1038/emboj.2012.99 · 10.75 Impact Factor
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ABSTRACT: Reporter gene assay is an invaluable tool for both biomedical and pharmaceutical researches to monitor cellular events associated with gene expression, regulation and signal transduction. On the basis of the alternations in reporter gene activities mediated by attaching response elements to these reporter genes, one sensitive, reliable and convenient assay can be provided to efficiently report the activation of particular messenger cascades and their effects on gene expression and regulations inside cells or living subjects. In this review, we introduce the current status of several commonly used reporter genes such as chloramphenicol acetyltransferase (CAT), alkaline phosphatase (AP), β-galactosidase (β-gal), luciferases, green fluorescent protein (GFP), and β-lactamase. Their applications in monitoring gene expression and regulations in vitro and in vivo will be summarized. With the development of advanced technology in gene expression and optical imaging modalities, reporter genes will become increasingly important in real-time detection of the gene expression at the single-cell level. This synergy will make it possible to understand the molecular basis of diseases, track the effectiveness of pharmaceuticals, monitor the response to therapies and evaluate the development process of new drugs.Biotechnology & genetic engineering reviews 01/2008; 25(1):41-75. DOI:10.5661/bger-25-41 · 1.90 Impact Factor