King S Thomas, Kenney Linda J
Department of Microbiology and Immunology, University of Immunology, University of Illinois-Chicago, Chicago, Illinois, USA.
Methods Enzymol. 2007;422:352-60. doi: 10.1016/S0076-6879(06)22017-2.
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.
EnvZ/OmpR双组分调节系统最为人所知的是，它能根据生长培养基渗透压的变化来调节孔蛋白基因ompF和ompC。响应未知信号时，EnvZ会被ATP在一个组氨酸残基上进行自身磷酸化。随后磷酸基团被转移到OmpR上一个保守的天冬氨酸残基上。OmpR的磷酸化增加了它对孔蛋白基因调节区域的亲和力，从而改变它们的表达。磷酸化还会改变EnvZ与OmpR之间的相互作用。为了研究EnvZ和OmpR的相互作用，我们采用了一种与绿色荧光蛋白（GFP）融合的全长EnvZ构建体，该构建体被过表达并定位于内膜。制备原生质球并在含有纯化的、荧光标记的OmpR蛋白的微量滴定板中裂解。发生了从GFP供体到荧光素或罗丹明偶联的OmpR受体的荧光共振能量转移（FRET），表明这两种蛋白相互作用。然后我们用FRET进一步表征磷酸化对相互作用参数的影响。结果表明，全长EnvZ的行为与单独的分离胞质结构域EnvZc相似。此外，磷酸化的OmpR蛋白对EnvZ激酶的亲和力降低。本章描述了关于此类实验的一般注意事项，并提供了定量测量它们的详细方案。
