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 kd pFABC operon, coding for the high affinity K+-translocating Kdp ATPase of Escherichia coil. The stimulus for KdpD to undergo autophosphoryla tion is believed to be a change in turgor or some effect thereof, refl ecting 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 t he C terminus. This KdpD-His(6), protein was functional in vitro and i n vivo, KdpD-His(6) was purified from everted membrane vesicles by sol ubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatogra phy to >99% homogeneity. The solubilized protein was not active with r espect to autophosphorylation, but retained the ability to bind a-azid o-ATP. KdpD-His(6), was reconstituted into proteoliposomes in a unidir ectional inside-out orientation as revealed by ATP accessibility and p rotease susceptibility. Purified and reconstituted KdpD-His(6), exhibi ted autokinase activity, and the phosphoryl group could be transferred to KdpE, Furthermore, KdpD-His(6), was found to be the only protein t hat mediates dephosphorylation of KdpE similar to P.