PMID:9099740
| Citation |
Jung, K, Tjaden, B and Altendorf, K (1997) Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli. J. Biol. Chem. 272:10847-52 |
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| Abstract |
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. |
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| Keywords |
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 |
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