ISSN 1007-7626 CN 11-3870 / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology 2015 10 31 10 1102 ~ 1108 DOI 10 13865 /j cnki cjbmb 2015 10 14 E coli Hfq * 130024 Hfq srna RNA Hfq mrna Hfq 1 1 Hfq Hfq 70% Hfq C Hfq Hfq 2 Hfq 1 1 63 Q5 Limited Proteolysis Improves E coli Hfq Crystal Structure Resolution FENG Shi-Qiong SI Yun-Long SONG Chen-Yang WANG Pei-Qi SU Ji-Yong * Department of Biochemistry and Molecular Biology School of Life Sciences Northeast Normal University Changchun 130024 China Abstract Hfq is an abundant RNA chaperone in bacteria Hfq facilitates srnas targeting their antisense RNAs that regulates bacteria responses in stressing environments Hfq is a homohexameric protein which has six histidine residues on the surface of its inner ring Therefore nickel affinity chromatography can be used to directly purify this protein from E coli After size exclusion chromatography the purity of this protein achieved 70% Before crystallization this protein was limited proteolysised by chymotrypsin to cut C-terminal flexible loop The digested Hfq fragment was crystallized in many conditions containing NH 4 2 SO 4 as precipitant But the initial needle-like crystals are too small to be used for crystal data collection After crystallization optimization bigger crystals grew in two conditions Finally two Hfq crystal structures were solved The resolution of one crystal structure was 1 63 Key words limited proteolysis protein crystal structure resolution protein purification protein crystallization optimization Hfq RNA srna mrna 1 Hfq srna mrna Qβ RNA Hfq 2-3 12 Hfq 4 2015-03-02 2015-04-21 Hfq σs σe 5-7 Hfq srna mrna mrna 8-11 Hfq srna mrna Hfq srna srna No 2412015KJ018 * Tel 0431-85099350 Fax 0431-85098212 E-mail sujy100@ nenu edu cn Received March 2 2015 Accepted April 21 2015 Surpported by the Fundamental Research Funds for the Central Universities No 2412015KJ018 * Corresponding author Tel 0431-85099350 Fax 0431-85098212 E-mail sujy100@ nenu edu cn
10 E coli Hfq 1103 Hfq Sm /Lsm 13-15 Hfq 1 N 1 α 5 β 1 1 5 β 2 Sm1 Sm2 16 6 Hfq Tris Hepes DTT Hfq 1 RNA Protease Inhibitor Cocktails Hfq 65 Sigma Amicon Ultra-15 30 kd 11 25 Hfq Millipore AmSO 4 Suite RNA Qiagen 96 Hampton proximal side distal side Research mrna srna KTApurifier 100 RNA 16-20 HiLoad 26 /600 Superdex 200 pg Hfq HiTrap IMAC GE 8 Coulter Dougals instrument 21 22 1 2 Hfq Rosseta TM 2 DE3 100 ml β α LB LB 50 μg /ml A 600 1 5 100 ml LB 21 23 10 L LB 37 6 000 g trypsin chymotrypsin Rosseta TM 2 DE3 Merck Emerson Bekerman 10 min 200 ml thermolysin V8 papain subtilisin 1 50 mmol /L Tris-HCl ph 8 0 300 mmol /L NaCl 20 mmol /L imidazole 24 21 20 000 g 30 min 5 24 1 PDB 2 50 mmol /L Tris-HCl ph 8 0 300 mmol /L Hfq NaCl 500 mmol /L imidazole Hfq Hfq 10 ml Hfq 3 10 mmol /L C Hfq Hepes ph 7 5 200 mmol /L NaCl 10% Glycerol 10 Hfq mmol /L DTT Hfq 55 kd Hfq Hfq 2 Hfq 1 63 1 98 Hfq KTApurifier 100 Hfq Hfq 5 mg ~ 20 mg /ml 15% SDS-PAGE Hfq
1104 31 1 3 Hfq 1 1 000 0 1 mol /L Tris-HCl ph Hfq 10 mg Hfq 8 0 1 4 mol /L NH 4 2 SO 4 15% glycerol 10 μg 80 1 h 20% glycerol 0 5 mol /L Li 2 SO 4 96 96 1 4 mol /L NH 4 2 SO 4 20% glycerol 50 μl Hfq Swiss light source X10SA PX II 1 h 1 1 0 2 μl 0 2 μl 96 25 1 5 3 X- 79 0 1 mol /L Tris-HCl ph 8 5 1 5 mol /L NH 4 2 SO 4 15% glycerol 80 0 5 mol /L Li 2 SO 4 1 6 mol /L NH 4 2 SO 4 Phaser 28 Hfq PDB Hfq 1HK9 molecular replacement Phenix 29 refine Hfq 1 4 79 Hfq X- XDS 25 integrate CCP4 26 SCALA 27 scale MolProbity 30-31 Hfq 2 Hfq PDB Hfq 2 Fig 1 Hfq purification and crystallization A Gel filtration and SDS-PAGE Two arrows indicated Hfq peak and Hfq protein band in gel filtration and SDS-PAGE respectively SDS-PAGE showed that the purity of Hfq protein was about 70% B and C Hfq crystals in two crystallization conditions The condition in B was 0 5 mol /L Li 2 SO 4 1 4 mol /L NH 4 2 SO 4 The condition in C was 0 1 mol /L Tris-HCl ph 8 0 1 4 mol /L NH 4 2 SO 4 15% glycerol The arrows indicated the Hfq needle crystals
10 E coli Hfq 1105 4RCB 4RCC Hfq 1 63 Table 2 1 Pymol 2 Li 2 SO 4 Hfq 1 2 1 Hfq 1 PDB 4RCB Gln 5 Hfq 6 His 71 2 PDB 4RCC Gln 5 Hfq 55 kd His 70 Hfq Hfq Hfq Fig 1A Hfq PDB 1HK9 Fig 2A Hfq 70% Hfq 1 α 5 β Fig 1A 2 2 Hfq 3 Hfq Table 1 79 80 Hfq Hfq 6 5 mg /ml 20 mg /ml Fig 2A 1 1 0 8 1 1 0 8 33 Hfq 4 25 37 Rosseta TM 2 DE3 1 μl 1 5 μl 2 μl 4 μl Rosseta TM 2 DE3 1 Hfq X- Hfq Fig 1B 1C 2 3 Hfq 0 5 mol /L Li 2 SO 4 Hfq 55 kd 1 L A 600 4 0 5mg Hfq Hfq 1 Table 1 The crystallization conditions of Hfq protein Number of AmSO 4 suite Components 1 2 2 mol /L Ammonium sulfate 3 0 2 mol /L Ammonium chloride 2 2 mol /L Ammonium sulfate 5 0 2 mol /L Ammonium fluoride 2 2 mol /L Ammonium sulfate 11 0 2 mol /L Ammonium tartrate 2 2 mol /L Ammonium sulfate 13 0 2 mol /L Cadmium sulfate 2 2 mol /L Ammonium sulfate 16 0 2 mol /L Ammonium bromide 2 2 mol /L Ammonium sulfate 18 0 2 mol /L Lithium chloride 2 2 mol /L Ammonium sulfate 23 0 2 mol /L Potassium bromide 2 2 mol /L Ammonium sulfate 24 0 2 mol /L Potassium chloride 2 2 mol /L Ammonium sulfate 32 0 2 mol /L K /Na tartrate 2 2 mol /L Ammonium sulfate 37 0 2 mol /L Sodium chloride 2 2 mol /L Ammonium sulfate 40 0 2 mol /L Sodium fluoride 2 2 mol /L Ammonium sulfate 41 0 2 mol /L Sodium formate 2 2 mol /L Ammonium sulfate 58 0 1 mol /L Hepes ph 7 0 1 6 mol /L Ammonium sulfate 59 0 1 mol /L Tris ph 8 0 1 6 mol /L Ammonium sulfate 60 0 1 mol /L Bicine ph 9 0 1 6 mol /L Ammonium sulfate 63 0 1 mol /L Mes ph 6 0 2 4 mol /L Ammonium sulfate 79 0 1 mol /L Tris ph 8 5 1 5 mol /L Ammonium sulfate 15% V /V Glycerol 80 0 5 mol /L Lithium chloride 1 6 mol /L Ammonium sulfate 82 0 2 mol /L Sodium chloride 0 1 mol /L Hepes ph 7 5 1 6 mol /L Ammonium sulfate 83 0 1 mol /L Hepes ph 7 5 1 6 mol /L Ammonium sulfate 2% m /V PEG1000 84 0 1 mol /L Mes ph 6 5 1 8 mol /L Ammonium sulfate
1106 31 Table 2 Crystal data collection and optimization of data statistics of the protein a Structure 1 Structure 2 Data collection temperature 100 K 100 K PDB number 4 RCB 4 RCC Wavelength 0 97159 0 97159 Resolution 28 16-1 63 30 66-1 98 Space group P6 P6 Unit cell parameters a b c 61 27 61 27 28 16 61 32 61 32 28 09 No of measured reflections 63 353 12 012 No of unique reflections 7 684 4 286 Completeness % 100 99 1 Multiplicity 8 2 2 8 Rmerge % b 0 3 0 1 < I /δ I > 5 8 6 0 Refinement Resolution limits 28 16-1 63 30 66-1 98 Rmodel % c 18 90 17 66 Rfree % d 19 23 20 52 No of waters 36 36 Rmsd bond lengths 0 0093 0 0084 Rmsd bond angles 1 253 1 288 Average B factor 22 1 25 0 Ramachandran plote residue in favored regions % 95 4 95 3 a Values in parentheses correspond to the highest-resolution shell b R merge = hkl i I hkl i -I hkl / hkl i I hkl i c R model = hkl F o hkl -F c hkl / hkl F o hkl where F o and F c are the observed and calculated structure factors respectively d A 5% random test set e Calculated using Molprobity Fig 2 Hfq structure analysis A The merged picture of three E coli Hfq crystal structures PDB 1HK9 4RCB 4RCC Cyanine indicated 1HK9 purple indicated 4RCB gold indicated 4RCC The N-and C-terminals of three crystal structures can not overlay with each other that means these two parts were flexible Six histidine residues in inner ring were close to each other that means Hfq protein can be directly purified from E coli crude extract by Ni-NTA B Proximal side and distal side of Hfq protein C The primary structure of E coli Hfq protein α-helix and β-sheet were indicated by blue and green respectively Arrows indicated predicative digestion site of E coli Hfq protein by chymotrypsin The proteinase prediction web site is http / /web expasy org peptide_cutter / Only His70 and His71 are visible in 4RCB and 4RCC that means chymotrypsin digestion sites are at His70 and His71
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