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

TY - JOUR

T1 - Demonstration of dimer formation of the cytoplasmic domain of a transmembrane osmosensor protein, EnvZ, of Escherichia coli using Ni-histidine tag affinity chromatography

AU - Hidaka, Yuji

AU - Park, Heiyoung

AU - Inouye, Masayori

N1 - Funding Information: We thank Ms. Linda Egger and Ms. Rinku Dutta for their critical reading of the manuscript. Y.H. thanks the Yamada Science foundation for a fellowship. This work was supported by Grant GM19043 from the National Institutes of Health.

PY - 1997/1/3

Y1 - 1997/1/3

N2 - 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 affnity 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.

AB - 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 affnity 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.

KW - Dimer formation

KW - EnvZ

KW - Histidine kinase

KW - OmpR

KW - Osmosensor

UR - https://www.scopus.com/pages/publications/0031022042

UR - https://www.scopus.com/pages/publications/0031022042#tab=citedBy

U2 - 10.1016/S0014-5793(96)01396-8

DO - 10.1016/S0014-5793(96)01396-8

M3 - Article

C2 - 9001405

SN - 0014-5793

VL - 400

SP - 238

EP - 242

JO - FEBS Letters

JF - FEBS Letters

IS - 2

ER -