Helical complexes formed between fd DNA and reductively methylated fd gene 5 protein were indistinguishable by electron microscopy from complexes formed with the nonmethylated protein. 13C NMR spectroscopy of 13C-enriched N epsilon, N epsilon-dimethyllsyl residues of the protein showed that three of these residues (Lys-24, Lys-46, and Lys-69) were selectively perturbed by binding of the oligomer d(pA)7. These were the same lysyl residues that we previously found to be most protected from methylation by binding of the protein to poly[r(U)] [Dick, L. R., Sherry, A. D., Newkirk, M. M., & Gray D. M. (1988) J. Biol. Chem. 263, 18864-18872]. Thus, these lysines are probably directly involved in the nucleic acid binding function of the protein. Negatively charged chelates of lanthanide ions were used to perturb the 13C NMR resonances of labeled lysyl and amino-terminal residues of the gene 5 protein. The terbium chelate was found to bind tightly (Ka approximately 10(5) M-1) to the protein with a stoichiometry of 1 chelate molecule per protein dimer. 13C resonances of Lys-24, Lys-46, and Lys-69 were maximally shifted by the terbium chelate and were maximally relaxed by the gadolinium chelate. Also, the terbium chelate was excluded by the oligomer d(pA)7. Computer fits of the induced chemical shifts of 13C resonances with those expected for various positions of the terbium chelate failed to yield a possible chelate binding site unless the chemical shift for Lys-24 was excluded from the fitting process.(ABSTRACT TRUNCATED AT 250 WORDS)