PMID:10026245
| Citation |
Racher, KI, Voegele, RT, Marshall, EV, Culham, DE, Wood, JM, Jung, H, Bacon, M, Cairns, MT, Ferguson, SM, Liang, WJ, Henderson, PJ, White, G and Hallett, FR (1999) Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts. Biochemistry 38:1676-84 |
|---|---|
| Abstract |
The ProP protein of Escherichia coli is an osmoregulatory H+-compatible solute cotransporter. ProP is activated by an osmotic upshift in both whole cells and membrane vesicles. We are using biochemical and biophysical techniques to explore the osmosensory and catalytic mechanisms of ProP. We now report the purification and reconstitution of the active transporter. Protein purification was facilitated by the addition of six histidine (His) codons to the 3' end of proP. The recombinant gene was overexpressed from the E. coli galP promoter, and ProP-(His)6 was shown to be functionally equivalent to wild-type ProP by enzymatic assay of whole cells. ProP-(His)6, purified by Ni2+ (NTA) affinity chromatography, cross-reacted with antibodies raised against the ProP protein. ProP-(His)6 was reconstituted into Triton X-100 destabilized liposomes prepared with E. coli phospholipid. The reconstituted transporter mediated proline accumulation only if (1) a membrane potential was generated by valinomycin-mediated K+ efflux and (2) the proteoliposomes were subjected to an osmotic upshift (0.6 M sucrose). Activity was also stimulated by DeltapH. Pure ProP acts, in the proteoliposome environment, as sensor, transducer, and respondent to a hyperosmotic shift. It is the first such osmosensor to be isolated. |
| Links |
PubMed Online version:10.1021/bi981279n |
| Keywords |
Bacterial Proteins/biosynthesis; Bacterial Proteins/genetics; Bacterial Proteins/isolation & purification; Bacterial Proteins/metabolism; Biological Transport; Carrier Proteins/biosynthesis; Carrier Proteins/genetics; Carrier Proteins/isolation & purification; Carrier Proteins/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins; Genetic Vectors/metabolism; Histidine/genetics; Kinetics; Osmolar Concentration; Proteolipids/metabolism; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Symporters |
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