Isolation and characterization of the amino and carboxyl proximal fragments of the adenosine cyclic 3' ,5'-phosphate receptor protein of Escherichia coli.
The cyclic AMP receptor protein (CRP) is a positive and negative regulatory protein for gene expression in Escherichia coli. The protein has been cleaved proteolytically to determine the relation between CRP structure and function. In the presence of sodium dodecyl sulfate (NaDodSO4), chymotrypsin dissects CRP into two stable fragments of molecular weight 9500 (9.5K) and 13 000 (13K). After removal of NaDodSO4, the two fragments are resolved by Bio-Rex 70 chromatography in 6 M urea. Analyses of the terminal amino acids released from each fragment and cyanogen bromide cleavage products indicate that the 9.5K fragment is amino proximal in CRP while the 13K fragment is carboxyl proximal. Notable features of amino acid composition are the relatively high amount of arginine and methionine in the 13K fragment and the retention in the 9.5K fragment of the two tryptophans present in the CRP subunit. Following isoelectric focusing in 8 M urea, the 9.5K fragment, 22.5K CRP, and 13K fragment migrate to pH 5.5, 8.3, and 10.3, respectively. While CRP is a cAMP-stimulated DNA binding protein, the 13K fragment binds to DNA in the presence and absence of cAMP. The 9.5K fragment associates to form dimers and decamers. These data are consonant with a model in which the DNA binding domain is present in the carboxyl proximal region of CRP while the amino proximal region contains the subunit-subunit interaction sites and much of the cAMP binding domain.
D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D000242:Cyclic AMP / Q000302:isolation & purification* / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011067:Poly dA-dT / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D010446:Peptide Fragments; D011067:Poly dA-dT; D000242:Cyclic AMP; D002918:Chymotrypsin; D003488:Cyanogen Bromide