13C NMR of Methylated Lysines of fd Gene 5 Protein: Evidence for a Conformational Change Involving Lysine 24 upon Binding of a Negatively Charged Lanthanide Chelate

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^ϵ,N^ϵ-dimethyllysyl 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 (K_α ≈ 10⁵ 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. With this exclusion, a possible chelate binding site was found to be a hydrophobic pocket centered between the two DNA-binding loops of the protein dimer. Chelate-induced shifts that could be measured for ¹H resonances agreed with this location of the binding site. All of our data would be consistent with this location if there were a substantial movement of the flexible DNA-binding loops containing Lys-24 upon binding of the chelate.