Expression, purification, mass spectrometry, crystallization and multiwavelength anomalous diffraction of selenomethionyl PvuII DNA methyltransferase (cytosine-N4-specific).
The type II DNA-methyltransferase (cytosine N4-specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14. Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine. Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two-column chromatography procedure. The yield of purified protein was approximately 1.8 mg/g bacterial paste. Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation. Amino acid sequencing and mass spectrometry analysis of selenomethionine-containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine. Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form. Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S-adenosyl-L-methionine in the monoclinic space group P2(1). Two complexes were present per asymmetric unit. Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.
O'Gara
M
M
W.M. Keck Structural Biology Laboratory, Cold Spring Harbor Laboratory, NY, USA.
Adams
G M
GM
Gong
W
W
Kobayashi
R
R
Blumenthal
R M
RM
Cheng
X
X
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; C047084:DNA modification methylase PvuII; D015257:DNA-Cytosine Methylases