Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni‐histidine tag affinity chromatography

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Abstract
EnvZ is a transmembrane osmosensor which regulates the phosphorylation of OmpR, a transcription factor for ompF and ompC genes which encode the major outer membrane porin proteins, OmpF and OmpC in Escherichia coli. Autophosphorylation of EnvZ occurs through a transphosphorylation reaction between two EnvZ molecules. To elucidate the molecular mechanism of signal transduction by EnvZ, we examined the dimer formation of the EnvZ cytoplasmic domain [EnvZ(C)]. For this purpose, we developed a method to determine the complex formation between the purified EnvZ(C) and the purified His6‐EnvZ(C) by means of Ni‐6xhistidine tag affinity chromatography. When the mixture of EnvZ(C) and His6‐EnvZ(C) was applied to Ni‐NTA resin, both His6‐EnvZ(C) and EnvZ(C) were bound to the resin, indicating that EnvZ can form an oligomer without the periplasmic and transmembrane domains. Binding experiments using the Ni‐NTA resin revealed that EnvZ(C) forms a dimer with the K a value for dimerization being approximately 105 M−1 in the equilibrium state.