Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni-histidine tag affinity chromatography.
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.
Hidaka
Y
Y
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Park
H
H
Inouye
M
M
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D003593:Cytoplasm; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression; D006639:Histidine; D009097:Multienzyme Complexes; D009532:Nickel; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012116:Resins, Plant / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D009942:Organometallic Compounds; D011993:Recombinant Fusion Proteins; D012116:Resins, Plant; C088321:nickel nitrilotriacetic acid; D006639:Histidine; D009532:Nickel; C057843:envZ protein, E coli; D009571:Nitrilotriacetic Acid