Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli.
The methyl-accepting chemotactic transducers of Escherichia coli were found to bind strongly to Cibacron blue-Sepharose. Among potential elutants tested, only S-adenosylmethionine at moderate concentrations and NaCl at concentrations greater than 1.5 M caused dissociation of these detergent-solubilized transmembrane proteins from the dye. Release by S-adenosylmethionine may be a generalized effect rather than the result of a specific binding site for that compound on transducers. A truncated trg gene was created that coded for the carboxyl-terminal three-fifths of the transducer, which constitutes the cytoplasmic domain common to all four transducers in E. coli. This domain bound to Cibacron blue-Sepharose and was eluted in a pattern similar to that exhibited by intact Trg, indicating that interaction with the dye occurred in this conserved domain. Adherence to Cibacron blue and elution by high salt formed the core of an efficient purification scheme, developed for Trg but applicable to all transducers in E. coli and perhaps to methyl-accepting chemotaxis proteins in other species. Determination of the amino acid sequence at the beginning of purified Trg confirmed that it contained a longer hydrophilic segment at its amino terminus than other transducers of E. coli. The initial methionine of Trg is neither cleaved nor modified, in contrast to the Tar transducer in which the amino terminus was found previously to be blocked. Circular dichroic measurements of purified Trg indicated that the secondary structural organization of the protein is predominantly alpha-helix.
Burrows
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Biochemistry/Biophysics Program, Washington State University, Pullman 99164-4660.
Newcomer
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ME
Hazelbauer
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GL
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000302:isolation & purification*; D002633:Chemotaxis; D004396:Coloring Agents; D003593:Cytoplasm / Q000032:analysis; D004926:Escherichia coli / Q000032:analysis*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008970:Molecular Weight; D011487:Protein Conformation; D012436:S-Adenosylmethionine; D012685:Sepharose / Q000031:analogs & derivatives
D000596:Amino Acids; D001426:Bacterial Proteins; D004396:Coloring Agents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C041476:Trg protein, E coli; C017842:cibacron blue-sepharose; D012436:S-Adenosylmethionine; D012685:Sepharose