Switching DNA-binding specificity by unnatural amino acid substitution

The specificity of protein-nucleic acid recognition is believed to originate largely from hydrogen bonding between protein polar atoms, primarily side-chain and polar atoms of nucleic acid bases. One way to design new nucleic acid binding proteins of novel specificity is by structure-guided alterations of the hydrogen bonding patterns of a nucleic acid-protein complex. We have used cI repressor of bacteriophage lambda as a model system. In the lambda-repressor-DNA complex, the epsilon-NH(2) group (hydrogen bond donor) of lysine-4 of lambda-repressor forms hydrogen bonds with the amide carbonyl atom of asparagine-55 (acceptor) and the O6 (acceptor) of CG6 of operator site O(L)1. Substitution of lysine-4 (two donors) by iso-steric S-(2-hydroxyethyl)-cysteine (one donor and one acceptor), by site-directed mutagenesis and chemical modification, leads to switch of binding specificity of lambda-repressor from C:G to T:A at position 6 of O(L)1. This suggests that unnatural amino acid substitutions could be a simple way of generating nucleic acid binding proteins of altered specificity.