Crystallization and preliminary X-ray crystallographic studies of glutaredoxin 2 from Saccharomyces cerevisiae in different oxidation states.
Glutaredoxins are small (9-12 kDa) heat-stable proteins that are highly conserved throughout evolution; the glutaredoxin active site (Cys-Pro-Tyr-Cys) is conserved in most species. Five glutaredoxin genes have been identified in Saccharomyces cerevisiae; however, Grx2 is responsible for the majority of oxidoreductase activity in the cell, suggesting that its primary function may be the detoxification of mixed disulfides generated by reactive oxygen species (ROS). Recombinant Grx2 was expressed in Escherichia coli as a 6xHis-tagged fusion protein and purified by nickel-affinity chromatography. Prior to crystallization trials, the enzyme was submitted to various treatments with reducing agents and peroxides. Crystals suitable for X-ray diffraction experiments were obtained from untreated protein and protein oxidized with t-butyl hydroperoxide (10 mM). Complete data sets were collected to resolutions 2.15 and 2.05 A for untreated and oxidized Grx2, respectively, using a synchrotron-radiation source. The crystals belong to space group P4(1)2(1)2, with similar unit-cell parameters.
Discola
Karen Fulan
KF
Departamento de Biologia, Instituto de Biociências, Universidade de São Paulo, Rua do Matão 277, São Paulo, SP, Brazil.
Oliveira
Marcos Antonio de
MA
Silva
Gustavo Monteiro
GM
Barcena
José Antonio
JA
Porras
Pablo
P
Padilla
Alicia
A
Netto
Luis Eduardo Soares
LE
Guimarães
Beatriz Gomes
BG
D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D054477:Glutaredoxins; D006639:Histidine / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014961:X-Ray Diffraction; D020122:tert-Butylhydroperoxide
D054477:Glutaredoxins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D020122:tert-Butylhydroperoxide; D010088:Oxidoreductases