J. Biol. Chem., Vol. 263, Issue 35, 18864-18872, Dec, 1988
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
LR Dick, AD Sherry, MM Newkirk and DM Gray
Program in Molecular and Cell Biology, University of Texas at Dallas, Richardson.
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).
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