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Bioinformaticians + Experimentalists = Successful Protein Analysis

Bioinformaticians + Experimentalists = Successful Protein Analysis. Katarzyna Poleszak. International Institute of Molecular and Cell Biology Laboratory of Bioinformatics and Protein Engineering. Research subjects. Enzymes acting on:. DNA. RNA. Discovering novel enzymes

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Bioinformaticians + Experimentalists = Successful Protein Analysis

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  1. Bioinformaticians + Experimentalists = Successful Protein Analysis Katarzyna Poleszak International Institute of Molecular and Cell Biology Laboratory of Bioinformatics and Protein Engineering

  2. Research subjects Enzymes acting on: DNA RNA • Discovering novel enzymes • Protein engineering • Characterization of protein complexes

  3. Structure of the laboratory Computer analysis Structure/function prediction Experimental validation

  4. What information do we get from protein structure? • Protein function • Biological processes • Mechanism of reaction and interaction

  5. Discovering newRNA methyltransferases Elżbieta Purta

  6. Protein synthesis in bacteria Amino end Growing polypeptide Amino acid 50S rRNA: 5S rRNA 23S rRNA tRNA mRNA 30S rRNA: 16S rRNA

  7. Modifying enzyme unknown Modifying enzyme identified in this lab Modified nucleosides in rRNA • 11 modified nucleosides in 16S; 26 in 23S • Main function: reinforce the tertiary structure essential for catalysis • Other functions: resistance to antibiotics that bind to rRNA

  8. YbeA: novelMTase specific for 23S rRNA Mass spectrometry analysis +CH3 (14Da)

  9. Interaction of YbeAwiththeribosome Katarzyna Kamińska

  10. Protein engineering of restriction endonucleases Sebastian Pawlak

  11. dsDNA REase AACTGGCCTGCCA TTCACCGGACGGT cleavage CCTGCCA GGACGGT AACTGG TTCACC cleaved DNA Restriction endonucleases (REases) • occur frequently in bacteria and archaea • cleave double-stranded DNA in a sequence-specific manner

  12. various cleaved sequences 266 distinct specificities ~ 3700 REases REBASE 2008 Why engineer REases? Important tools in biology: recombinant DNA technology diagnostic tool DNA physical mapping

  13. 4000 3500 3000 2500 liczba specyficzności 2000 number of specificities REases liczba ER 1500 1000 500 0 1985 1990 1995 2000 2007 1980 1970 1975 Increase in amount of discovered REases versus specificities

  14. ? structure unknown Engineering of restriction endonuclease Bsp6I Bsp6I 5' GCNGC CGNCG 5' N = C, G, A, T

  15. Bsp6I model dr Janusz Bujnicki

  16. Predicted effect of Bsp6I mutagenesis No contact with middle bases G C N G C C G N C G G C N G C C G N C G Creating contact with middle bases E94 K

  17. Bsp6I with novel specificity GCNGC - + GCWGC GCSGC GCGGC GCAGC GCTGC GCCGC Activity [%] Activity [%] wt wt/R94K wt wt/R94K

  18. Characterization of protein complexes involved in DNA repair Katarzyna Poleszak

  19. Why study protein complexes involved in DNA repair? Protein-protein interactions Protein interaction sites DNA repair Processes correcting DNA damages Crucial to maintain genome stability Critical to most biological processes Potential drug targets

  20. hMLH1 MBD4 MUTATION Hereditary nonpolyposis colorectal cancer(HNPCC) Analysing hMLH1-MBD4 interactions ? humanMutLhomolog Methyl binding domain

  21. Functions of MBD4 Methyl binding domain MBD4 Binds methylated DNA Tumor supresor T/U glycosylase

  22. hMLH1 Functions of hMLH1 humanMutLhomolog Depending on the interaction partner Corects base-base mismatches Recombination Removes insertions and deletions

  23. Yeast two-hybrid system (Y2H) - +

  24. Screening protein-protein interactions Cotransformation Leu MBD-AD hMLH1-BD Trp Strong interactions (HIS, ADE) Weak interactions (HIS) Medium without Leu, Trp, His, Ade Medium without Leu, Trp Medium without Leu, Trp, His

  25. Prediction of hMLH1 protein interaction sites Jan Kosiński

  26. 1 498 In domain Ex domain Ex domain hMLH1 humanMutLhomolog NTD N – terminal domain CTD C – terminal domain 756 • highly conserved • less conserved • involved in • homo-, and heterodimerization

  27. 1 498 1 498 756 499 555 686 756 1 498 569 677 1 498 756 499 555 569 677 686 756 hMLH1 – MBD4 Y2H 756 + wt hMLH1 - +/- - + - 498 756 499 555 686 756 +/- 569 677

  28. hMLH1 mutations L574P P640S P648L P648S Jan Kosiński

  29. Measuring the strength of interaction ONPG ᵦ-gal ᵦ-gal ONPG ortho-nitrophenol+ galactose

  30. hMLH1 mutants-MBD4 Relative strength of interaction ᵦ-galactosidase [U] Control L574P E578G P640S Y646C P648L P648S hMLH1 mutants

  31. Mutations abolishing hMLH1-MBD4 interaction

  32. Summary Bioinformaticians + Experimentalists= Successful Protein Analysis

  33. www.genesilico.pl

  34. Acknowledgements Phd Janusz Bujnicki Phd Krzysztof Skowronek Elżbieta Purta Sebastian Pawlak Katarzyna Kamińska Jan Kosiński

  35. Thank you for your attention

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