Bloconlugate Chem. 1003, 4, 219-225
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N-(Iodoacetyl)-p-phenylenediamine-EDTA: A Reagent for High-Efficiency Incorporation of an EDTA-Metal Complex at a Rationally Selected Site within a Protein Yon W. Ebright,? Yan Chen,t Richard D. Ludescher,* and Richard H. Ebright'st Department of Chemistry and Waksman Institute, and Department of Food Science, Rutgers University, New Brunswick, New Jersey 08855. Received December 10, 1992 ~~
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We have developed a highly efficient procedure to incorporate an EDTA:metal complex at a rationally selected site within a full-length protein. Our procedure has two steps: In step one, we use site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest. In step two, we derivatize the resulting protein with N-(iodoacety1)-p-phenylenediamine-EDTAmetal,a novel haloacetyl derivative of EDTA:metal. We have used this procedure to incorporate each of three EDTA metal complexes at amino acid 2 of the helix-turn-helix motif of the sequence-specific DNA binding protein Cro: a radioactive and nucleolytic EDTAmetal complex (EDTA55Fe),a radioactive EDTA metal complex (EDTA:63Ni),and a fluorescent and heavy-atom EDTA:metal complex (EDTAEu). Incorporation of EDTAmetal was highly efficient (>80% for EDTA55Fe and EDTAFNi; 60% for EDTAEu) and highly site-specific(>99% ). We have analyzed DNA affinity cleavingby the Cro derivative having EDTA:55Feat amino acid 2 of the helix-turn-helix motif. The Cro derivative cleaves DNA at base pairs -4 to 6 of the DNA half site in the protein-DNA complex, indicating that amino acid 2 of the helix-turn-helix motif of Cro is close to base pairs -4 to 6 of the DNA half site in the Cro-DNA complex in solution. The results are in agreement with the crystallographic structure of the Cro-DNA complex [Brennan, R., Roderick, S., Takeda, Y., and Matthews, B. (1990) h o c . Natl. Acad. Sci. U.S.A. 87,81651 and with a previous DNA affinity cleaving analysis of the Cro-DNA complex [Ebright, Y., Chen, Y., Pendergrast, P. S., and Ebright, R. (1992) Biochem. 31,106641. The procedure of this report has potential applications in DNA affinity cleaving with sequence-specific DNA binding proteins, sitespecific radioactive labeling of protein, site-specific fluorescent labeling of protein, and site-specific heavy-atom labeling of protein.
EDTA forms complexes with a wide range of metal ions, including radioactive, fluorescent, and reactive metal ions (1, 2). EDTAmetal complexes can be conjugated to proteins having specificity for target biomolecules [e.g., monoclonal antibodies, sequence-specific DNA binding proteins, etc. (3-27)l. The resulting (EDTA:metal)protein conjugates have applications as radiodiagnostics, radiotherapeutics, and affinity cleaving reagents. For certain applications, it is essential to incorporate an EDTAmetal complex at arationally selected site within a protein. One such application is EDTAFe DNA affinity cleaving to determine the location and orientation of the DNA site for a sequence-specific DNA binding protein (14,16-18,23,24,26). 1nEDTA:FeDNA affiinitycleaving, one incorporates EDTA:Fe at an amino acid not critical for protein-DNA complexformation but nevertheless close to DNA in the protein-DNA complex, forms the derivatized protein-DNA complex,initiates EDTAFe-mediated DNA cleavage by addition of reducing agent, and determines the nucleotide(s) a t which EDTAFe-mediated DNA cleavage occurs. Previously, incorporation of an EDTAmetal complex at a rationally selected site within a protein has been possible only by (i) total synthesis of the protein (14-18, 23-25) or (ii) site-directed mutagenesis to introduce a unique solvent-accessible cysteine residue at the site of interest followed by derivatization of the resulting protein with S-(2-pyridylthio)cysteamine-EDTAmeta11, an aro-
* Author to whom correspondence should be addressed. Telephone: (908) 932-5179. Telefax: (908) 932-5735. + Department of Chemistry and Waksman Institute. Department of Food Science.
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1043-1802/93/2904-0219$04.00/0
matic disulfide derivative of EDTA:metal(26; cf. ref 27). Procedure i is limited to small proteins (typically
