PMID:9001405
| Citation |
Hidaka, Y, Park, H and Inouye, M (1997) Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni-histidine tag affinity chromatography. FEBS Lett. 400:238-42 |
|---|---|
| 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 Ka value for dimerization being approximately 10(5) M(-1) in the equilibrium state. |
| Links | |
| Keywords |
Bacterial Outer Membrane Proteins/chemistry; Bacterial Outer Membrane Proteins/genetics; Bacterial Outer Membrane Proteins/isolation & purification; Chromatography, Affinity; Cytoplasm; Escherichia coli/metabolism; Escherichia coli Proteins; Gene Expression; Histidine; Multienzyme Complexes; Nickel; Nitrilotriacetic Acid/analogs & derivatives; Nitrilotriacetic Acid/chemistry; Organometallic Compounds/chemistry; Recombinant Fusion Proteins/chemistry; Recombinant Fusion Proteins/genetics; Recombinant Fusion Proteins/isolation & purification; Resins, Plant/chemistry |
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