Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli.
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.
Jung
K
K
Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Universität Osnabrück, D-49069 Osnabrück, Federal Republic of Germany.
Tjaden
B
B
Altendorf
K
K
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003470:Culture Media; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D009876:Operon; D009994:Osmolar Concentration; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011188:Potassium / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D015398:Signal Transduction
D001386:Azides; D001426:Bacterial Proteins; D003470:Culture Media; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; C489580:KdpD protein, E coli; D011494:Protein Kinases; C075616:kdpD protein, Bacteria; D011188:Potassium