Identification of lysine residues at the binding site of bacteriophage-Pf1 DNA-binding protein.
The accessibility of NH2 groups in the DNA-binding protein of Pf1 bacteriophage has been investigated by differential chemical modification with the reagent ethyl acetimidate. The DNA-binding surface was mapped by identification of NH2 groups protected from modification when the protein is bound to bacteriophage-Pf1 DNA in the native nucleoprotein complex and when bound to the synthetic oligonucleotide d(GCGTTGCG). The ability of the modified protein to bind to DNA was monitored by fluorescence spectroscopy. Modification of the NH2 groups in the native nucleoprotein complex showed that seven out of the eight lysine residues present, and the N-terminus, were accessible to the reagent, and were not protected by DNA or by adjacent protein subunits. Modification of these residues did not inhibit the ability of the protein to bind DNA. Lysine-25 was identified by peptide mapping as being the major protected residue. Modification of this residue does abolish DNA-binding activity. Chemical modification of the accessible NH2 groups in the complex formed with the octanucleotide effectively abolishes binding to DNA. Peptide mapping established that, in this case, lysine-17 was the major protected residue. The differences observed in protection from acetimidation, and in the ability of the modified protein to bind DNA, indicate that the oligonucleotide mode of binding is not identical with that found in the native nucleoprotein complex with bacteriophage-Pf1 DNA.
D000596:Amino Acids / Q000032:analysis; D001435:Bacteriophages / Q000378:metabolism*; D001665:Binding Sites; D002850:Chromatography, Gel; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008239:Lysine / Q000032:analysis*; D046911:Macromolecular Substances; D009844:Oligosaccharides; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
D000596:Amino Acids; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D010446:Peptide Fragments; D008239:Lysine