Computational design of DNA-binding proteins

Kono, Hidetoshi; Imanishi, Miki*; Negi, Shigeru*; Tatsutani, Kazuya*; Sugiura, Yukio*

We have computationally designed proteins with zinc-finger motifs and experimentally characterized them, successfully obtaining DNA-binding proteins with high DNA sequence specificity. Given a backbone structure, we calculated amino acid sequences which are compatible with the backbone so that they can fold into an expected structure at room temperature. We then tried to add a function of DNA-binding to the amino acid sequences by reconsidering a part of the sequences composing of the DNA recognition helix. The sequences for the helix were computationally selected that had the lowest protein-DNA interaction energy for the target DNA sequence. The designed proteins were structurally characterized by CD spectrum and functionally examined by gel shift assay. More than 60% of experimentally tested proteins showed the helix induction upon the addition of zinc ions. This indicates that the designed proteins at least have the expected secondary structure. Among the designed proteins, one showed a higher DNA sequence-specificity than the natural protein whose backbone structure was used as template in design. We demonstrate that our computational approach is efficient to design proteins that bind to a specific DNA sequence.