Isolation and characterization of the amino and carboxyl proximal fragments of the adenosine cyclic 3',5'-phosphate receptor protein of Escherichia coli

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Abstract
The cAMP receptor protein (CRP) is a positive and negative regulatory protein for gene expression in E. coli. The protein was cleaved proteolytically to determine the relation between CRP structure and function. In the presence of sodium dodecyl sulfate (NaDodSO4), chymotrypsin dissects CRP into 2 stable fragments of MW 9500 (9.5K) and 13,000 (13K). After removal of NaDodSO4, the 2 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 2 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. The data support 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.