Production and properties of the α core derived from the cyclic adenosine monophosphate receptor protein of Escherichia coli

Abstract

Proteolytic digestion of the cyclic adenosine monophosphate (cAMP) receptor protein (CRP) in the presence of cAMP results in the formation of a resistant core molecule. The CRP core retains the dimeric structure and cAMPbinding properties of the native CRP, while cAMP-dependent DNA binding is lost. CRP consists of two identical polypeptides of molecular weight 22 500. The CRP cores formed after proteolytic digestion in the presence of 0.1 mM cAMP differ in molecular weight: subtilisin and trypsin CRP cores, ~12 500; chymotrypsin CRP core, ~13 000; and Straphylococcus V8 protease CRP core, ~18 000. In the absence of cAMP, CRP is relatively resistant to these enzymes. The aCRP core produced by subtilisin digestion has been characterized as follows: The sedimentation coefficient of the aCRP 1 ranscription of catabolite-repressible operons involves the cAMP-dependent binding of CRP' to a site within the promoter of these operons (Pastan and Adhya, 1976). Proteolysis of the cyclic AMP receptor protein (CRP) in the presence of cAMP produces the a core fragment (aCRP) whose subunit of ~12 500 molecular weight is derived from the native CRP subunit of 22 500 molecular weight (Krakow and Pastan, 1973). While the CRP shows cAMP binding and cAMPdependent DNA binding at pH 8, the a core does not show this activity at pH 8, although its cAMP binding activity is retained. We have previously shown that cAMP produces a conformational change in the a core, resulting in an increased resistance to proteolysis and denaturation (Eilen and Krakow, 1977a). This event, in the native CRP, has been postulated to function as a conformational signal in effecting the formation of the DNA-binding site in the /3 region of the protein (Eilen f From the

A characterization by sequencing of the termini of the polypeptide chain of cyclic AMP receptor protein from Escherichia coli and the subtilisin produced N‐terminal fragment

“…The protein has M, 45 000 and it is composed of 2 apparently identical subunits [ll]. In the presence of CAMP limited proteolytic digestion of CRP with subtilisin results in a resistant N-terminal core, a-CRP, which retains the dimeric structure and CAMP binding activity but has lost CAMP-dependent DNA-binding capacity at pH 8 as judged by the nitrocellulose filter assay [12,13]. However, from a thermal denaturation study on (u-CRP was prepared essentially as in [ 131 in the following way.…”

DNA binding of cAMP receptor protein and its N‐terminal core stabilizes the double helix and is modulated by the allosteric effector cAMP

“…This model was suggested on the basis of computer modelling on the interactions between DNA and the polypeptide acarbon backbone of CBP, based on the 2.9 A resolution crystal structure of the CAMP -CBP complex [8]. If this hypothesis is correct, the N-terminal core of CBP, aCBP, produced by subtilisin digestion of the CAMP -CBP complex, in which the small carboxy-terminal domain has been removed [9], should not bind to DNA. Electron microscopy has shown that the binding of CBP non-specifically to DNA results in the formation of a complex with regular striations along the DNA whose length is -&times shorter than that of free DNA [lo].…”

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

“…The sequence also contains several stretches that resemble the CRP-binding site in the gal operon (10). Also available are the proteins involved in the regulation of the ara operon: araC protein (11), CRP (12), and RNA polymerase (13).…”

“…CRP was purified as described (12), and araC protein was purified as described (11), with the substitution of a hydroxyapatite column for the DEAE-Sephadex column. It was loaded in 0.014 M potassium phosphate buffer at pH 6.9, 10% (vol/vol) glycerol, 0.01 M L-arabinose, 1 mM EDTA, and 1 mM dithiothreitol and eluted in the same buffer with the phosphate raised to 0.13 M.…”