DNA sequence selectivity and kinetic properties of de novo designed metalloprotein dimers

Abstract

In our efforts to engineer a DNA binding and cleaving protein with greater sequence discrimination, we have designed dimeric proteins derived from engrailed homeodomain and calmodulin. Previous research by our group has shown that a hydrolytically active lanthanide binding site can be incorporated into a DNA binding motif. To understand protein-DNA interaction and improve the sequence selectivity of the chimeric complex, two lanthanide-binding homodimers were designed and expressed. One of the dimers, F2, is coupled together by a flexible polypeptide linker and the other, R7C, is a disulfide cross-linked cysteine mutant at the N-terminus. Studies of fluorescence of tryptophan residues document that the overall affinity for lanthanide and calcium is similar to traditional EF-hand peptides (1-10 µM). Metal titrations monitored by circular dichroism (CD) revealed that the secondary structures of the dimers contained a lower degree of α-helicity than the designed monomeric protein due to additional modifications, but because of their flexibility and their two active-site domain, hydrolytic activity was several folds faster than our previously designed proteins and peptides. Unlike earlier reports on our chimeras, F2 also demonstrated the capability to hydrolyze DNA in the presence of some biological relevant metal ions suggesting different cleavage mechanisms were carried out. Extensive DNA sequencing studies on cleavage patterns with oligonucleotide duplexes confirmed the unique sequence selectivity and kinetic properties of F2. Two engrailed homeodomain target sites, TAATTA, were favored for hydrolytic activity corresponding to one domain acting as a DNA anchor on the first target site while the other was an "opportunist" at recognizing the second site. Nonetheless, the hydrolytic behavior at the phosphodiester bond on a specific dsDNA sequence is in good agreement with the behavior of restriction endonucleases. Unlike restriction enzymes, metallated F2 has not only demonstrated the ability to cleave DNA plasmid, but it also excises the entire nucleotide on a selected sequence. This homodimer v Caroline and Alida showed me how to be "kick-ass" as a woman both literally and theoretically. They have become my second family whom I will always cherish. At home, I would like to thank you for the encouragement and perseverance of my aunt, Auntie Goat, who is no doubt the inspiration for my success nowadays. Nonetheless, no one else can compare to my devoting and loving parents, Kin Sang Lau and Cheuk Yut Wong. I would not be the person I am now without them as they have given up so much in order to provide me the best quality of life. They have showed me to be strong through tough times and celebrate the happiness in life. Last but not the least, I feel absolutely lucky that I have met my husband, Stephen Deyrup, who is my best friend and my companion in life. We have gone through many huddles together throughout all these years and his unconditional love, support, and trust for me remain untainted. His smile is my sunsh...