Purification and characterization of the periplasmic domain of the aspartate chemoreceptor.
In order to facilitate biochemical studies of cell-surface receptors, a plasmid allowing the expression of the periplasmic domain of the aspartate receptor from Salmonella typhimurium as a soluble periplasmic protein has been constructed. This 18-kDa protein is exported to the periplasm, where it may be extracted by mild osmotic lysis. This isolated domain behaves as a normal, soluble protein and has been purified to homogeneity by standard techniques. The purified periplasmic domain binds aspartate with a kD similar to that of the full-length receptor, and the binding occurs with negative cooperativity, i.e. the binding of one molecule of aspartate induces a conformational change that interferes with the binding of the second aspartate. Unlike the full-length receptor, the periplasmic domain undergoes a protein concentration- and aspartate-dependent monomer-dimer equilibrium. At low protein concentrations and in the absence of aspartate, the protein is monomeric. At higher protein concentrations or in the presence of saturating aspartate, the protein is dimeric. Two charge variants of the protein have been identified on native polyacrylamide gels. The more acidic form is blocked to Edman degradation, indicating that modification of the amino terminus of this protein can occur after cleavage of the signal peptide in the periplasm.
Milligan
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Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Koshland
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D001224:Aspartic Acid / Q000378:metabolism*; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D017459:Receptors, Amino Acid / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012486:Salmonella typhimurium / Q000378:metabolism*
D006882:Hydroxyapatites; D017459:Receptors, Amino Acid; D011994:Recombinant Proteins; C083267:aspartic acid receptor, Salmonella typhimurium; D001224:Aspartic Acid; D017886:Durapatite