ISSN CN / Q http / /cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology ~ 1108 DOI

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1 ISSN CN / Q http / /cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 1108 DOI /j cnki cjbmb E coli Hfq * Hfq srna RNA Hfq mrna Hfq 1 1 Hfq Hfq 70% Hfq C Hfq Hfq 2 Hfq 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 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 Hfq Hfq σs σe 5-7 Hfq srna mrna mrna 8-11 Hfq srna mrna Hfq srna srna No KJ018 * Tel Fax sujy100@ nenu edu cn Received March Accepted April Surpported by the Fundamental Research Funds for the Central Universities No KJ018 * Corresponding author Tel Fax sujy100@ nenu edu cn

2 10 E coli Hfq 1103 Hfq Sm /Lsm Hfq 1 N 1 α 5 β β 2 Sm1 Sm Hfq Tris Hepes DTT Hfq 1 RNA Protease Inhibitor Cocktails Hfq 65 Sigma Amicon Ultra kd Hfq Millipore AmSO 4 Suite RNA Qiagen 96 Hampton proximal side distal side Research mrna srna KTApurifier 100 RNA HiLoad 26 /600 Superdex 200 pg Hfq HiTrap IMAC GE 8 Coulter Dougals instrument Hfq Rosseta TM 2 DE3 100 ml β α LB LB 50 μg /ml A ml LB L LB 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 mmol /L NaCl 20 mmol /L imidazole g 30 min PDB 2 50 mmol /L Tris-HCl ph mmol /L Hfq NaCl 500 mmol /L imidazole Hfq Hfq 10 ml Hfq 3 10 mmol /L C Hfq Hepes ph mmol /L NaCl 10% Glycerol 10 Hfq mmol /L DTT Hfq 55 kd Hfq Hfq 2 Hfq 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

3 Hfq mol /L Tris-HCl ph Hfq 10 mg Hfq mol /L NH 4 2 SO 4 15% glycerol 10 μg 80 1 h 20% glycerol 0 5 mol /L Li 2 SO mol /L NH 4 2 SO 4 20% glycerol 50 μl Hfq Swiss light source X10SA PX II 1 h μl 0 2 μl X mol /L Tris-HCl ph mol /L NH 4 2 SO 4 15% glycerol mol /L Li 2 SO mol /L NH 4 2 SO 4 Phaser 28 Hfq PDB Hfq 1HK9 molecular replacement Phenix 29 refine Hfq Hfq X- XDS 25 integrate CCP4 26 SCALA 27 scale MolProbity 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 mol /L NH 4 2 SO 4 The condition in C was 0 1 mol /L Tris-HCl ph mol /L NH 4 2 SO 4 15% glycerol The arrows indicated the Hfq needle crystals

4 10 E coli Hfq RCB 4RCC Hfq 1 63 Table 2 1 Pymol 2 Li 2 SO 4 Hfq 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 Hfq Hfq 6 5 mg /ml 20 mg /ml Fig 2A Hfq 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 mg Hfq Hfq 1 Table 1 The crystallization conditions of Hfq protein Number of AmSO 4 suite Components mol /L Ammonium sulfate mol /L Ammonium chloride 2 2 mol /L Ammonium sulfate mol /L Ammonium fluoride 2 2 mol /L Ammonium sulfate mol /L Ammonium tartrate 2 2 mol /L Ammonium sulfate mol /L Cadmium sulfate 2 2 mol /L Ammonium sulfate mol /L Ammonium bromide 2 2 mol /L Ammonium sulfate mol /L Lithium chloride 2 2 mol /L Ammonium sulfate mol /L Potassium bromide 2 2 mol /L Ammonium sulfate mol /L Potassium chloride 2 2 mol /L Ammonium sulfate mol /L K /Na tartrate 2 2 mol /L Ammonium sulfate mol /L Sodium chloride 2 2 mol /L Ammonium sulfate mol /L Sodium fluoride 2 2 mol /L Ammonium sulfate mol /L Sodium formate 2 2 mol /L Ammonium sulfate mol /L Hepes ph mol /L Ammonium sulfate mol /L Tris ph mol /L Ammonium sulfate mol /L Bicine ph mol /L Ammonium sulfate mol /L Mes ph mol /L Ammonium sulfate mol /L Tris ph mol /L Ammonium sulfate 15% V /V Glycerol mol /L Lithium chloride 1 6 mol /L Ammonium sulfate mol /L Sodium chloride 0 1 mol /L Hepes ph mol /L Ammonium sulfate mol /L Hepes ph mol /L Ammonium sulfate 2% m /V PEG mol /L Mes ph 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

5 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 Resolution Space group P6 P6 Unit cell parameters a b c No of measured reflections No of unique reflections Completeness % Multiplicity Rmerge % b < I /δ I > Refinement Resolution limits Rmodel % c Rfree % d No of waters Rmsd bond lengths Rmsd bond angles Average B factor Ramachandran plote residue in favored regions % 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

6 10 E coli Hfq 1107 srna RNA Hfq srna srna Hfq Fig 2B RNA Hfq Hfq 1HK HK9 N C Hfq N C 1 C β- Fig 2A Hfq J Genes Dev Hfq 70% Hfq Hfq Hfq Hfq C Fig 2C beta J Hfq 9 Azam TA Hiraga S Beich-Frandsen Hajnsdorf E Regnier P Hfq Hfq 2 85 Hfq Hfq C 73 ~ 102 Hfq P1 P6 Hfq C Hfq involved in Sm protein-protein interactions J References 1 RNA Hfq DsrA rpos 3462 Wang L J Wang W W Gong Q G et al Research progress of E coli RNA chaperone Hfq interaction with DsrA and rpos J Prog Biochem Biophys Franze de Fernandez M T Eoyang L August JT Factor fraction required for the synthesis of bacteriophage Qbeta-RNA J Nature Schuppli D Miranda G Tsui H C et al Altered 3'-terminal RNA structure in phage Qbeta adapted to host factor-less Escherichia coli J Proc Natl Acad Sci U S A Robinson KE Orans J Kovach AR et al Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching J Nucleic Acids Res Muffler A Fischer D Hengge-Aronis R The RNA-binding protein HF-I known as a host factor for phage Qbeta RNA replication is essential for rpos translation in Escherichia coli Guisbert E Rhodius VA Ahuja N et al Hfq modulates the sigmae-mediated envelope stress response and the sigma32- mediated cytoplasmic stress response in Escherichia coli J J Bacteriol Brown L Elliott T Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I an RNAbinding protein encoded by the hfq gene J J Bacteriol Kajitani M Kato A Wada A et al Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q J Bacteriol Ishihama A Two types of localization of the DNA-binding proteins within the Escherichia coli nucleoid J Genes Cells Host factor Hfq of Escherichia coli stimulates elongation of poly A tails by poly A polymerase I J Proc Natl Acad Sci U S A Vytvytska O Moll I Kaberdin VR et al Hfq HF1 stimulates ompa mrna decay by interfering with ribosome binding J Genes Dev srna Hfq Song L Zhou X L Yue J J et al srna chaperone Hfq in bacteria a research progress J Bull Acad Mil Med Sci Hermann H Fabrizio P Raker VA et al snrnp Sm proteins share two evolutionarily conserved sequence motifs which are EMBO J Seraphin B Sm and Sm-like proteins belong to a large family identification of proteins of the U6 as well as the U1 U2 U4 and U5 snrnps J EMBO J Salgado-Garrido J Bragado-Nilsson E Kandels-Lewis S et al Sm and Sm-like proteins assemble in two related complexes of deep evolutionary origin J EMBO J Schumacher MA Pearson RF Moller T et al Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex a bacterial Sm-like protein J EMBO J

7 Sauer E Weichenrieder O Structural basis for RNA 3'-end recognition by Hfq J Proc Natl Acad Sci U S A Link TM Valentin-Hansen P Brennan RG Structure of Escherichia coli Hfq bound to polyriboadenylate RNA J Proc Natl Acad Sci U S A Someya T Baba S Fujimoto M et al Crystal structure of Hfq from Bacillus subtilis in complex with SELEX-derived RNA aptamer insight into RNA-binding properties of bacterial Hfq J Nucleic Acids Res Murina V Lekontseva N Nikulin A Hfq binds ribonucleotides in three different RNA-binding sites J Acta Crystallogr D Biol Crystallogr Pt Fontana A de Laureto PP Spolaore B et al Probing protein structure by limited proteolysis J Acta Biochim Pol Hubbard SJ The structural aspects of limited proteolysis of native proteins J Biochim Biophys Acta Reed RG Feldhoff RC Peters T Jr Fragments of bovine serum albumin produced by limited proteolysis complementary behavior of two large fragments J Biochemistry Dong A Xu X Edwards AM et al In situ proteolysis for protein crystallization and structure determination J Nat Methods Kabsch W XDS J Acta Crystallogr D Biol Crystallogr Pt Potterton E Briggs P Turkenburg M et al A graphical user interface to the CCP4 program suite J Acta Crystallogr D Biol Crystallogr Pt Evans P Scaling and assessment of data quality J Acta Crystallogr D Biol Crystallogr Pt McCoy AJ Grosse-Kunstleve RW Adams PD et al Phaser crystallographic software J J Appl Crystallogr Pt Adams PD Afonine PV Bunkoczi G et al PHENIX a comprehensive Python-based system for macromolecular structure solution J Acta Crystallogr D Biol Crystallogr Pt Chen VB Arendall WB 3rd Headd JJ et al MolProbity allatom structure validation for macromolecular crystallography J Acta Crystallogr D Biol Crystallogr Pt Davis IW Leaver-Fay A Chen VB et al MolProbity all-atom contacts and structure validation for proteins and nucleic acids J Nucleic Acids Res Web Server issue W375- W Bolanos-Garcia VM Davies OR Structural analysis and classification of native proteins from E coli commonly co-purified by immobilised metal affinity chromatography J Biochim Biophys Acta Yonekura K Watanabe M Kageyama Y et al Posttranscriptional regulator Hfq binds catalase HPII crystal structure of the complex J PLoS One e Beich-Frandsen M Vecerek B Sjoblom B et al Structural analysis of full-length Hfq from Escherichia coli J Acta Crystallogr Sect F Struct Biol Cryst Commun Pt

第 33 卷第 2 期 2010 年 4 月 - 149 - 脱蛋白较好的方法是 Sevag 法, 它是根据蛋白质在氯仿等有机溶剂中变性的特点, 使蛋白质变性成胶状, 此法条件温和, 可避免多糖的降解

第 33 卷第 2 期 2010 年 4 月 - 149 - 脱蛋白较好的方法是 Sevag 法, 它是根据蛋白质在氯仿等有机溶剂中变性的特点, 使蛋白质变性成胶状, 此法条件温和, 可避免多糖的降解 - 148 - Drug Evaluation Research Vol.33 No.2 April 2010 植物多糖的研究进展陶遵威 1, 郑夺 1, 邸明磊 2, 单晓菊 1 天津市医药科学研究所, 天津 300020; 2 天津中医药大学, 天津 300073 2 摘要多糖是由单糖连接而成的多聚物,