Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli

J Biol Chem. 1997 Apr 18;272(16):10847-52. doi: 10.1074/jbc.272.16.10847.


In response to K+ availability or medium osmolality, the sensor kinase KdpD and the response regulator KdpE control the expression of the kdpFABC operon, coding for the high affinity K+-translocating Kdp ATPase of Escherichia coli. The stimulus for KdpD to undergo autophosphorylation is believed to be a change in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic osmotic solute. The membrane-bound sensor kinase KdpD was overproduced as a fusion protein containing six contiguous histidine residues two amino acids before the C terminus. This KdpD-His6 protein was functional in vitro and in vivo. KdpD-His6 was purified from everted membrane vesicles by solubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatography to >99% homogeneity. The solubilized protein was not active with respect to autophosphorylation, but retained the ability to bind 2-azido-ATP. KdpD-His6 was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His6 exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE. Furthermore, KdpD-His6 was found to be the only protein that mediates dephosphorylation of KdpE approximately P.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / analogs & derivatives
  • Adenosine Triphosphate / metabolism
  • Amino Acid Sequence
  • Azides / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism*
  • Culture Media
  • DNA Primers
  • Escherichia coli / genetics
  • Escherichia coli / physiology*
  • Escherichia coli Proteins*
  • Molecular Sequence Data
  • Operon
  • Osmolar Concentration
  • Phosphorylation
  • Polymerase Chain Reaction
  • Potassium / metabolism
  • Protein Kinases / chemistry*
  • Protein Kinases / isolation & purification
  • Protein Kinases / metabolism*
  • Protein Structure, Secondary*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Tagged Sites
  • Signal Transduction


  • Azides
  • Bacterial Proteins
  • Culture Media
  • DNA Primers
  • Escherichia coli Proteins
  • Recombinant Proteins
  • 2-azidoadenosine 5'-triphosphate
  • Adenosine Triphosphate
  • KdpD protein, E coli
  • Protein Kinases
  • kdpD protein, Bacteria
  • Potassium