Protein Engineering Protocols - Mycobacteriology research center

5Design and Synthesis of Artificial Zinc Finger ProteinsWataru Nomura and Yukio SugiuraSummaryOf the DNA-binding motifs, the (Cys) 2(His) 2-type zinc finger motif has great potential formanipulation. The zinc finger motif offers an attractive framework for the design of novel DNAbindingproteins. Specially, a structure-based design strategy is valuable for the creation of newartificial zinc finger proteins that have novel DNA-binding properties, namely, long-DNA recognition,DNA bending, and AT-rich sequence recognition. Herein, new strategies for the design ofmulti-zinc finger proteins for the recognition of a target DNA sequence, a DNA-bending zinc fingerprotein, a (His) 4-type zinc finger protein, and an AT-recognizing zinc finger protein aredescribed based on recent experimental results.Key Words: Zinc finger; multifinger; DNA bending; GC recognition; AT recognition;(Cys) 2(His) 2type; (His) 4type; artificial protein; helix substitution; protein design.1. IntroductionArtificial zinc finger proteins have important applications in biomedicalresearch and in gene therapy, and are novel tools for biochemical and molecularbiological investigations (1–3). In particular, a (Cys) 2(His) 2-type zinc fingermotif, one of the most common DNA-binding motifs in eukaryotes, presents aversatile and attractive framework for the design of new DNA-binding proteins.The structural analysis of some (Cys) 2(His) 2-type zinc finger protein–DNAcomplexes revealed the characteristics of this motif in DNA binding as follows:1. The zinc finger structure is repeated by the connection with a specific linker.2. Each zinc finger structure binds to a 3-basepair (bp) subsite of DNA with its α-helixfacing toward the major groove.3. The amino acid residues at four key positions in the α-helix of each zinc fingerunit make a 1:1 contact with the DNA bases at specific positions.4. The overall arrangement of the peptide is antiparallel to the primary interactingstrand of DNA (4–7).From: Methods in Molecular Biology, vol. 352: Protein Engineering ProtocolsEdited by: K. M. Arndt and K. M. Müller © Humana Press Inc., Totowa, NJ83