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Bioinformatics & Structural Biology

Bioinformatics & Structural Biology. M93360008 生技所 研一 劉怡萱. ☆ JBC 2004 Oct. Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidis in complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol.

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Bioinformatics & Structural Biology

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  1. Bioinformatics & Structural Biology M93360008 生技所 研一 劉怡萱

  2. ☆ JBC 2004 Oct. Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidisin complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol Jason Gunawan1, Dave Simard2, Michel Gilbert3, Andrew L. Lovering1, Warren W. Wakarchuk3,Martin E. Tanner2, Natalie C. Strynadka1

  3. Mass Spectrometry ☆ Analysis of Tertiary and Quaternary Structure of Sialic Acid Synthase by Chemical and MS method

  4. ☆ Sialic acid & sialic acid synthase “Characterization of Escherichia coli Sialic Acid Synthase” Biochem. Biophys. Rapid Comm.2002, 295, 16 • Sialic acids, a family of 3-deoxy-2-keto nine carbon sugars are located in the non-reducing terminal ends of cell-surface glycoconjugates of virus, mammalian cells, and some bacteria. • The gene neuB encodes the enzyme, sialic acid synthase, catalyzing the condensation of ManAc with PEP to give sialic acid. Streptococcus aglactiae

  5. sialic acid synthase Product

  6. ☆ Evidence of Dimeric Dimer DMA 8.6 Å DMS 11.0 Å DMA : Dimethyl adipimidate DMS : Dimethyl suberimidate·2HCl SDS-PAGE

  7. Introduction to Peptide Mass Fingerprinting In solution digestion In gel digestion peptide fragments intact protein proteolysis MALDI-TOF MS Analyze MVYI I AE I GCNHNGDINLAKKMVDVAVSCGVDAVKFQTFK AEKLISKFAPKAEYQKATTGTADSQLEMTKRLELSFEEYLE MRDYAISKGVETFSTPFDEESLEFLISTDMPIYKIPSGE ITNLPYLEKLGKQQKKVILSTGMAVMEEIHQAVNILRQNG TTDISILHCTTEYPTPYPSLNLNVIHTLKDEFKDLTIGYS DHSIGSEVPIAAAAMGAEVIEKHFTLDTNMEGPDHKASAT PDILAALVKGVRIVEQALGRFEKIPDPVEEKNKIVARKSV VALKPIKKGDIYSIENITVKRPGNGISPMNWYDILGQEAQ DDFEEDEVIRDSRFENQLPELHHHHHH Database search

  8. ☆ The time resolved limited proteolysis and differential MALDI-TOF MS mapping In solution tryptic digestion 5min MALDI- MS M/Z 10min MALDI- MS GDIYSIENITVK M/Z 20min MALDI- MS DEESLEFLISTDMPIYK ITNLPYLEK M/Z

  9. ☆ The time resolved limited proteolysis and differential MALDI-TOF MS mapping 347 334 116 288 N 89 223 157 5min 10min 20min 30min Over night

  10. m/z ☆Identification of Cross-Linked Peptides in the Interface Regions Tryptic digestion Surface-labeled peptides Intramolecular cross-linked peptides SUBTRACT Unmodified peptides Putative intermolecular cross-linked peptides Intensity Chemical cleavage Intensity m/z

  11. ☆ Chemical cross-linking by EGS M.W 456.36 Spacer Arm 16.1Ǻ + -NH2 -NH2 COMPLEX EGS ( a NHS ester) Crosslinking +228 +245 -NH -NH -NH -1 -1 -1 INTERMOLECULAR + 226 Da -NH HYDROLYSED + 244 Da -NH INTRAMOLECULAR + 226 Da - - - + +82Da +82Da +82Da +164Da

  12. ☆ Mapping interface peptides by MALDI-TOF Unmodification peptide X Hydrolysed peptide Intermolecular cross-linked peptide Relative intensity(%) Intramolecular cross-linked peptide Cleaved fragments of intermolecular cross-linked peptide Cleaved fragments of intramolecular cross-linked peptide

  13. ☆ Proposed Interface Regions of NeuB 261-273 44-51 278-287 57-71 347C N 347C N 36-51 57-71 116-132 261-277 278-287 256 NeuB domain Proposed interface Antifreeze-like Domain

  14. ☆ JBC 2004 Oct. Structural and mechanistic analysis of sialic acid synthase NeuB from Neisseria meningitidisin complex with Mn2+, phosphoenolpyruvate, and N-acetylmannosaminitol Jason Gunawan1, Dave Simard2, Michel Gilbert3, Andrew L. Lovering1, Warren W. Wakarchuk3,Martin E. Tanner2, Natalie C. Strynadka1

  15. ☆ The eight-stranded a /b barrel (TIM barrel)

  16. ☆ The importance of ligands

  17. ☆ Multiple Sequence Alignments of NeuB family

  18. ☆ Multiple Sequence Alignments of NeuB from Neisseria Meningitidis and Streptococcus aglactiae SaNeuB 1 ------------------MVYIIAEIGCNHNGDINLAKKMVDVAVSCGVDAVKFQTFKAEKLISKFAPKAN.NeuB 1 MQNNNEFKIGNRSVGYNHEPLIICEIGINHEGSLKTAFEMVDAAYNAGAEVVKHQTHIVEDEMSDEAKQVSaNeuB 53 EYQKATTGTADSQLEMTKRLELSFEEYLEMRDYAISKGVETFSTPFDEESLEFLISTDMPIYKIPSGEITN.NeuB 71 IPGNADV----SIYEIMERCALNEEDEIKLKEYVESKGMIFISTPFSRAAALRLQRMDIPAYKIGSGECNSaNeuB 123 NLPYLEKLGKQQKKVILSTGMAVMEEIHQAVNILRQNGTTDISILHCTTEYPTPYPSLNLNVIHTLKDEFN.NeuB 137 NYPLIKLVASFGKPIILSTGMNSIESIKKSVEIIREAG-VPYALLHCTNIYPTPYEDVRLGGMNDLSEAFSaNeuB 193 KDLTIGYSDHSIGSEVPIAAAAMGAEVIEKHFTLDTNMEGPDHKASATPDILAALVKGVRIVEQALGRFEN.NeuB 206 PDAIIGLSDHTLDNYACLGAVALGGSILERHFTDRMDRPGPDIVCSMNPDTFKELKQGAHALKLARGGKKSaNeuB 263 KIPDPVEEKNKIVARKSVVALKPIKKGDIYSIENITVKRPGNGISPMNWYDILG-QEAQDDFEEDEVIRDN.NeuB 276 DTIIAGEKPTKDFAFASVVADKDIKKGELLSGDNLWVKRPGNGDFSVNEYETLFGKVAACNIRKGAQIKKSaNeuB 332 SRFENQLPELN.NeuB 346 TDIE------ ☆ the sturcture of S.NeuB sould be similar to N.NeuB

  19. ☆ Homology modelong of Sa NeuB

  20. ☆ The time resolved limited proteolysis and differential MALDI-TOF MS mapping 347 334 116 288 N 89 223 157 5min 10min 20min 30min Over night

  21. 347 334 116 288 N 89 223 157

  22. 347 334 116 288 N 89 223 157

  23. 347 334 116 288 N 89 223 157

  24. 44-51 57-71 261-273 278-287 347C N 347C N 36-51 57-71 116-132 261-277 278-287 A subunit 44-51 B subunit 36-51 B subunit 57-71

  25. 44-51 57-71 261-273 278-287 347C N 347C N 36-51 57-71 116-132 261-277 278-287 A subunit 116-132 B subunit 57-71

  26. 44-51 57-71 261-273 278-287 347C N 347C N 36-51 57-71 116-132 261-277 278-287 A subunit 278-287 B subunit 116-132

  27. A subunit 261-277 B subunit 278-287 A subunit 278-287 B subunit 261-277 44-51 57-71 261-273 278-287 347C N 347C N 36-51 57-71 116-132 261-277 278-287

  28. H8 H7 H7 H8

  29. H7 H8

  30. ☆Conclusion ☆ the sturcture of S.NeuB sould be similar to N.NeuB ☆ the imperfection of the method of “ time resolved limited proteolysis” ☆ does the tetramer real exist ?

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