Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate

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Abstract
Photoaffinity labeling of the c[cyclic]AMP receptor protein (CRP) of E. coli with 8-azidoAMP (8-N3cAMP) was demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analog. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5''AMP. Apparently (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, 1/3 remains associated with the amino-proximal .alpha. core region of CRP which contains the cAMP binding domain; the remaining 2/3 of the label associated with the .beta. region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the MW 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. A part of the carboxyl-proximal region may be folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.