Solubility engineering of the HhaI methyltransferase.
DNA methylation is involved in epigenetic control of numerous cellular processes in eukaryotes, however, many mechanistic aspects of this phenomenon are not yet understood. A bacterial prototype cytosine-C5 methyltransferase, M.HhaI, serves as a paradigm system for structural and mechanistic studies of biological DNA methylation, but further analysis of the 37 kDa protein is hampered by its insufficient solubility (0.15 mM). To overcome this problem, three hydrophobic patches on the surface of M.HhaI that are not involved in substrate interactions were subjected to site-specific mutagenesis. Residues M51 or V213 were substituted by polar amino acids of a similar size, and/or the C-terminal tetrapeptide FKPY was replaced by a single glycine residue (Delta324G). Two out of six mutants, delta324G and V213S/delta324G, showed improved solubility in initial analyses and were purified to homogeneity using a newly developed procedure. Biochemical studies of the engineered methyltransferases showed that the deletion mutant delta324G retained identical DNA binding, base flipping and catalytic properties as the wild-type enzyme. In contrast, the engineered enzyme showed (i) a significantly increased solubility (>0.35 mM), (ii) high-quality 2D-[(15)N,(1)H] TROSY NMR spectra, and (iii) (15)N spin relaxation times evidencing the presence of a monomeric well-folded protein in solution.
Daujotyte
Dalia
D
Laboratory of Biological DNA Modification, Institute of Biotechnology, LT-2028 Vilnius, Lithuania.
Vilkaitis
Giedrius
G
Manelyte
Laura
L
Skalicky
Jack
J
Szyperski
Thomas
T
Klimasauskas
Saulius
S
D019943:Amino Acid Substitution; D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008745:Methylation; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D013050:Spectrometry, Fluorescence
C053100:DNA modification methylase HinfI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)