Incorporation of an EDTA-Metal Complex at a Rationally Selected Site within a Protein: Application to EDTA-Iron DNA Affinity Cleaving with Catabolite Gene Activator Protein (CAP) and Cro

We have developed a simple procedure to incorporate an EDTA-metal complex at a rationally selected site within a full-length protein. Our procedure has two, steps: In step 1, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step 2, we derivatize the resulting protein with S-(2-pyridylthio)cysteamiriyl-EDTA-metal, a novel aromatic disulfide derivative of EDTA-metal. We have used this procedure to incorporate an EDTA-iron complex at amino acid 2 of the helix-turn-helix motif of each of two helix-turn-helix motif sequence-specific DNA binding proteins, catabolite gene activator protein (CAP) and Cro, and we have analyzed EDTA-iron-mediated DNA affinity cleavage by the resulting protein derivatives. The CAP derivative cleaves DNA at base pair 2 of the DNA half-site in the protein-DNA complex, and the Cro derivative cleaves DNA at base pairs-3 to 5 of the DNA half-site in the protein-DNA complex. We infer that amino acid 2 of the helix-turn-helix motif of CAP is close to base pair 2 of the DNA half-site in the CAP-DNA complex in solution and that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs-3 to 5 of the DNA half-site in the Cro-DNA complex in solution. The results are in excellent agreement with the crystallographic structures of the CAP-DNA and Cro-DNA complexes [Schultz, S., Shields, S., & Steitz, T. (1991) Science 253, 1001; Brennan, R., Roderick, S., Takeda, Y., & Matthews, B. (1990) Proc. Natl. Acad. Sci. US.A. 87, 8165]. In addition to EDTA-iron affinity cleaving with sequence-specific DNA binding proteins, the procedure of this report has potential applications in site-specific radioactive labeling of protein, site-specific fluorescent labeling of protein, and site-specific heavy-atom labeling of protein.