Reductive methylation and 13C NMR studies of the lysyl residues of fd gene 5 protein. Lysines 24, 46, and 69 may be involved in nucleic acid binding.
We have examined the role of lysyl residues in the binding of fd gene 5 protein to a nucleic acid polymer. The lysyl residues of the protein were chemically modified to form N epsilon, N epsilon-dimethyllysyl derivatives containing 13C-enriched methyl groups. The 13C NMR spectrum of the modified protein was studied as a function of pH and salt concentration. Differences in the local magnetic environment of the six dimethyllysyl amino groups allowed all six 13C resonances to be resolved for samples in the pH range 8.5-9.0 at less than 50 mM ionic strength. One of the dimethylamino resonances was split at low pH, indicating that the two methyl groups were nonequivalent and that the corresponding lysyl residue (either Lys-3 or Lys-7) might be involved in an ion-pairing interaction. Specific lysyl residues were protected from methylation when the protein was bound to poly(rU). The level of protection of individual lysyl residues was quantitated using peptide mapping and sequencing of gene 5 protein labeled with 3H and 14C radioactive labels. Lysines 24, 46, and 69 showed significant protection (33-52%) from methylation in the protein-polynucleotide complex, suggesting that these 3 residues form part of the nucleic acid-binding site. The alpha-amino group of Met-1 was relatively unreactive in both the free and bound protein, which indicated that the amino terminus is not as exposed in solution as in the crystal structure (Brayer, G.D., and McPherson, A. (1983) J. Mol. Biol. 169, 565-596).
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D006863:Hydrogen-Ion Concentration; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D014764:Viral Proteins; C057816:gene 5 protein, Enterobacteria phage M13; D004247:DNA; D008239:Lysine