Crystallization and preliminary X-ray diffraction analysis of Q4DV70 from Trypanosoma cruzi, a hypothetical protein with a putative thioredoxin domain.
Q4DV70 is annotated in the Trypanosoma cruzi CL Brener genome as a hypothetical protein with a predicted thioredoxin-like fold, although the catalytic cysteine residues that are conserved in typical oxidoreductases are replaced by serine residues. Gene-expression analysis indicates that this protein is differentially expressed during the T. cruzi life cycle, suggesting that it plays an important role during T. cruzi development. The gene coding for Q4DV70 was cloned and the protein was overexpressed in Escherichia coli with an N-terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method. A diffraction data set was collected to 1.50 A resolution from a single crystal grown in 25% PEG 1500, 200 mM sodium thiocyanate pH 6.9, 10 mM phenol and 10% ethylene glycol. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.04, b = 50.32, c = 61.18 A. The Q4DV70 structure was solved by molecular replacement using protein disulfide isomerase from yeast (PDB code 2b5e) as a search model. Initial refinement of the model indicated that the solution was correct. These data are being used for refinement of the model of Q4DV70.
dos Santos
Camila Ramos
CR
Center for Structural Molecular Biology (CeBiME), Brazilian Synchrotron Light Laboratory (LNLS), CP 6192, 13084-971 Campinas-SP, Brazil.
Fessel
Melissa Regina
MR
Vieira
Leandro de Carvalho
Lde C
Krieger
Marco Aurélio
MA
Goldenberg
Samuel
S
Guimarães
Beatriz Gomes
BG
Zanchin
Nilson Ivo Tonin
NI
Barbosa
João Alexandre Ribeiro Gonçalves
JA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D003460:Crystallization; D003625:Data Collection; D004926:Escherichia coli / Q000235:genetics; D017125:Genes, Protozoan; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D017510:Protein Folding; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D013223:Statistics as Topic; D013879:Thioredoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D014169:Transformation, Bacterial; D014349:Trypanosoma cruzi / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006639:Histidine; D013879:Thioredoxins