N-(iodoacetyl)-p-phenylenediamine-EDTA: a reagent for high-efficiency incorporation of an EDTA-metal complex at a rationally selected site within a protein.
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 derivatized the resulting protein with N-(iodoacetyl)-p-phenylenediamine-EDTA:metal, 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 EDTA:metal complex (EDTA:55Fe), a radioactive EDTA:metal complex (EDTA:63Ni), and a fluorescent and heavy-atom EDTA:metal complex (EDTA:Eu). Incorporation of EDTA:metal was highly efficient (> 80% for EDTA:55Fe and EDTA:63Ni; 60% for EDTA:Eu) and highly site-specific (> 99%). We have analyzed DNA affinity cleaving by the Cro derivative having EDTA:55Fe at 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Ebright
Y W
YW
Department of Chemistry, Rutgers University, New Brunswick, New Jersey 08855.
Chen
Y
Y
Ludescher
R D
RD
Ebright
R H
RH
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry*; D005063:Europium / Q000737:chemistry; D007504:Iron Radioisotopes / Q000737:chemistry; D007553:Isotope Labeling / Q000379:methods; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D009532:Nickel / Q000737:chemistry; D009690:Nucleic Acid Conformation; D010655:Phenylenediamines / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011868:Radioisotopes / Q000737:chemistry; D012997:Solvents; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004279:DNA, Viral; D007504:Iron Radioisotopes; D008670:Metals; C082057:N-(iodoacetyl)-4-phenylenediamine-EDTA; D010655:Phenylenediamines; D011506:Proteins; D011868:Radioisotopes; D012997:Solvents; D013438:Sulfhydryl Compounds; D005063:Europium; D009532:Nickel; D004492:Edetic Acid; D003545:Cysteine