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

Autor(en): Jung, K
Tjaden, B
Altendorf, K 
Stichwörter: 2 REGULATORY COMPONENTS; Biochemistry & Molecular Biology; CONTROL EXPRESSION; DRIVEN POTASSIUM-TRANSPORT; KDPABC OPERON; KINASE-ACTIVITY; OSMOSENSOR; PHOSPHORYLATION; SIGNAL TRANSDUCTION; STRUCTURAL PROTEINS; SYSTEM
Erscheinungsdatum: 1997
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 272
Ausgabe: 16
Startseite: 10847
Seitenende: 10852
Zusammenfassung: 
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-His(6), protein was functional in vitro and in vivo, KdpD-His(6) 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 a-azido-ATP. KdpD-His(6), was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His(6), exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE, Furthermore, KdpD-His(6), was found to be the only protein that mediates dephosphorylation of KdpE similar to P.
ISSN: 00219258

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