title: Characterization and directed evolution of a methyl-binding domain protein for high-sensitivity DNA methylation analysis.
abstract: Methyl-binding domain (MBD) family proteins specifically bind double-stranded, methylated DNA which makes them useful for DNA methylation analysis. We displayed three of the core members MBD1, MBD2 and MBD4 on the surface of Saccharomyces cerevisiae cells. Using the yeast display platform, we determined the equilibrium dissociation constant of human MBD2 (hMBD2) to be 5.9 ± 1.3 nM for binding to singly methylated DNA. The measured affinity for DNA with two methylated sites varied with the distance between the sites. We further used the yeast display platform to evolve the hMBD2 protein for improved binding affinity. Affecting five amino acid substitutions doubled the affinity of the wild-type protein to 3.1 ± 1.0 nM. The most prevalent of these mutations, K161R, occurs away from the DNA-binding site and bridges the N- and C-termini of the protein by forming a new hydrogen bond. The F208Y and L170R mutations added new non-covalent interactions with the bound DNA strand. We finally concatenated the high-affinity MBD variant and expressed it in Escherichia coli as a green fluorescent protein fusion. Concatenating the protein from 1× to 3× improved binding 6-fold for an interfacial binding application.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Building E19-502C, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Heimer
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Building E19-502C, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Brooke E
authors.initials: BE
authors.lastname: Tam
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Building E19-502C, 77 Massachusetts Avenue, Cambridge, MA 02139, USA sikes@mit.edu.
authors.forename: Hadley D
authors.initials: HD
authors.lastname: Sikes
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution / Q000379:methods*; D057890:Epigenomics / Q000379:methods*; D004926:Escherichia coli; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment
chemical_list: D004268:DNA-Binding Proteins
keywords: DNA methylation; directed evolution; epigenotyping; high affinity; methyl-binding domain protein