Protein crystallization and preliminary determination of three-dimensional structure of rice B-glucosidase (original) (raw)

Abstract

Rice Os4BGlu12, a glycosyl hydrolase family 1 (GH1) β-glucosidase hydrolyzes β-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the β-(1,3)linked disaccharide laminaribiose. Os4BGlu12 was expressed as an N-terminal thioredoxin/His 6 fusion protein in OrigamiB(DE3) Escherichia coli. The fusion protein was purified by immobilized metal-affinity chromatography (IMAC) with cobalt resin. After the thioredoxin/His 6 tag was excised from the protein with enterokinase, the fusion tag was removed by adsorption to the IMAC resin, which yielded a 55 kDa Os4BGlu12 with >95% purity. The free Os4BGlu12 enzyme and a complex with 2,4-dinitrophenyl 2-fluoro-2-deoxy-β-D-glucopyranoside (G2F) inhibitor were crystallized at 15°C. Microbatch crystallization screening generated the native crystals in 25% (w/v) polyethylene glycol (PEG) 4000, 0.1 M Tris-HCl, pH 8.5, 0.20 M NaCl. The conditions were further optimized by the hanging-drop vapordiffusion method with microseeding. Native crystals and crystals of Os4BGlu12 complexed with G2F were obtained in 19% (w/v) PEG 3350, 0.1 M Tris-HCl, pH 8.5, 0.16 M NaCl and 19% (w/v) PEG 2000, 0.1 M Tris-HCl, pH 8.5, 0.16 M NaCl, respectively. Crystals of free Os4BGlu12 and Os4BGlu12-G2F complex were diffracted to 2.50 and 2.45 Å resolution, respectively, and their unit cell symmetry VII determined to be in the tetragonal P4 3 2 1 2 space group. The structure of native Os4BGlu12 was solved by molecular replacement with the 1CBG structure as a search model and had two molecules per asymmetric unit with a solvent content of 49.98% and a Mathews Coefficient (V M ) of 2.46 Å 3 Da -1 . The native Os4BGlu12 structure further served as a template for rigid body refinement to solve the Os4BGlu12 with G2F data set, which had a V M of 2.68 Å 3 Da -1 and 54.17% solvent content. The structures were similar to previous known GH1 enzymes, but the significant differences were seen at the main-chain trace of loop surrounding the active site. The active site is located at the bottom of an approximately 20 Å deep slot-like pocket surrounded by a large surface loop. In the innermost part of the active site in the crystal structure of G2F complex, the surrounding conserved amino acid residues seen in other GH1 enzymes formed hydrogen bonds with the glucosyl unit.

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References (99)

  1. II MATERIALS AND METHODS .........................................................................17
  2. 1 Materials..........................................................................................................17
  3. 1.1 Recombinant plasmid............................................................................17 2.1.2 Chemicals and Reagents .......................................................................17
  4. 2 General methods..............................................................................................18 2.2.1 Transformation and of recombinant expression plasmid into expression host cells................................................................................................18
  5. 2.2 Expression of recombinant β-glucosidase was produced as thioredodoxin-Os4BGlu12 fusion protein in E.Coli system.................19
  6. 2.3 Protein extraction from E.Coli..............................................................19 2.2.4 Purification of recombinant thioredodoxin-Os4BGlu12 nonfusion protein ...................................................................................................20
  7. 2.5 Purification of recombinant-Os4BGlu12 nonfusion protein.................20
  8. 2.6 Protein analysis .....................................................................................21 2.2.6.1 SDS-PAGE electrophoresis ......................................................21
  9. 2.6.2 Enzyme activity ........................................................................22 2.2.6.3 Bio-Rad protein assay ...............................................................23
  10. Protein Crystallization.....................................................................................23 2.3.1 Initial screening for crystallization conditions......................................23 3.2.4 Optimization of Os4BGlu12-G2F complex crystal ..............................49
  11. 3 Crystal characterization...................................................................................50
  12. Preliminary X-ray diffraction analysis of native Os4BGlu12 and complex Os4BGlu12 crystals ........................................................................................51
  13. 5 Molecular replacement ....................................................................................53
  14. 6 Structure determination of free-Os4BGlu12 and Os4BGlu12 with G2F inhibitor...........................................................................................................54
  15. 6.1 The model quality of freeOs4BGlu12 and G2F complex.....................54
  16. 6.2 The Overall structure of Os4BGlu12 with and without G2F .......................................................................................................57
  17. 6.3 Comparison of Os4BGlu12 structure with other known GH1 structures .....................................................................................61
  18. 6.4 Enzyme active site ................................................................................66
  19. IV DISCUSSION ........................................................................................................75 4.1 Protein expression and purification.................................................................75 4.2 Crystallization of Os4BGlu12 crystal .............................................................76
  20. 2.1 Initial crystallization .............................................................................76
  21. 2.2 Optimization of crystal .........................................................................77
  22. 3 Crystal structure of Os4BGlu12 ......................................................................79
  23. 3.1 Overall structure....................................................................................79 1, 19%PEG 2000, 0.16 M NaCl 7, 19%PEG 3350, 0.16 M NaCl 2, 19%PEG 2000, 0.18 M NaCl 8, 19%PEG 3350, 0.18 M NaCl 3, 19%PEG 2000, 0.20 M NaCl 9, 19%PEG 3350, 0.20 M NaCl 4, 19%PEG 2000, 0.22 M NaCl 10, 19%PEG 3350, 0.22 M NaCl 5, 19%PEG 2000, 0.24 M NaCl 11, 19%PEG 3350, 0.24 M NaCl 6, 19%PEG 2000, 0.26 M NaCl 12, 19%PEG 3350, 0.26 M NaCl All crystals shown in Figure 3.12 were diffracted at the NSRRC, Taiwan. The cryoprotectant soaking conditions of the crystals for X-ray diffraction were optimized by varying the concentrations of precipitant and glycerol, and the incubation temperature (room temperature and 15°C). Some obtained crystals were incubated in various cryoprotectant solution overnight and the crystal surfaces were observed (Figure 3.12). The best condition was obtained when the crystal was soaked in the cryoprotectant solution containing 28% glycerol and a precipitant solution concentration increased by 22% and performed at 15°C. The soaked crystals were flash-frozen in the liquid nitrogen for 15 second. The crystals in conditions number 1 and 8 (Figure 3.12) were soaked in the cryoprotectant solution containing 2 mM 2,4- dinitrophenyl 2-fluoro-2-deoxy-β-D-glucopyranoside (G2F) inhibitor and 20 mM cellotetraose, respectively. Then the crystal was mounted in a cryo-loop and was placed onto goniometer head between the X-ray beam and the detector. The diffraction data were processed with the HKL2000 package. The crystals soaked with G2F and with cellotetraose were diffracted to 2.90 Å and 2.80 Å resolutions, respectively. Only one free Os4BGlu12 crystal obtained from 19% PEG 3350, 0.1 M Tris-HCl, pH 8.5, and 0.16 M NaCl gave 2.50 Å resolution. reflections of 39533 and 43131 and R sym values of 10.9% and 9.4%, respectively. The diffraction data of the Os4BGlu12 crystal with C4 and Os4BGlu12-G2F complex crystal that grew in 0.18 M NaCl were collected to completeness 99.9% the resolution ranges of 30.0-2.65 Å and 30.0-2.80 Å, which correlated with low numbers of unique reflections of 34255 and 29438, respectively. The four data sets have two molecules per asymmetric unit. The Matthews coefficients (V M ), were calculated to be 2.46 Å 3
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