Bioinformaticians Experimentalists = Successful Protein Analysis

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Bioinformaticians Experimentalists
Successful Protein Analysis

• Katarzyna Poleszak

International Institute of Molecular and Cell
Biology Laboratory of Bioinformatics and
Protein Engineering
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Research subjects
Enzymes acting on
DNA
RNA

• Discovering novel enzymes
• Protein engineering
• Characterization of protein complexes

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Structure of the laboratory
Computer analysis
Structure/function prediction
Experimental validation
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What information do we get from protein
structure?

• Protein function
• Biological processes
• Mechanism of reaction and interaction

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Discovering newRNA methyltransferases
Elzbieta Purta
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Protein synthesis in bacteria
50S rRNA 5S rRNA 23S rRNA
30S rRNA 16S rRNA
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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

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YbeA novel MTase specific for 23S rRNA
Mass spectrometry analysis
CH3 (14Da)
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Interaction of YbeA with the ribosome
Katarzyna Kaminska
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Protein engineering of restriction endonucleases
Sebastian Pawlak
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Restriction endonucleases (REases)

• occur frequently in bacteria and archaea
• cleave double-stranded DNA in a sequence-specific
  manner

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Why engineer REases?
Important tools in biology
recombinant DNA technology
diagnostic tool
DNA physical mapping
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Increase in amount of discovered REases versus
specificities
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Engineering of restriction endonuclease Bsp6I
Bsp6I
5'
GCNGC CGNCG
5'
N C, G, A, T
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Bsp6I model
dr Janusz Bujnicki
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Predicted effect of Bsp6I mutagenesis
No contact with middle bases
Creating contact with middle bases
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Bsp6I with novel specificity
GCNGC
-

Activity
Activity
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Characterization of protein complexes involved
in DNA repair
Katarzyna Poleszak
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Why study protein complexes involved in DNA
repair?
DNA repair
Processes correcting DNA damages
Crucial to maintain genome stability

• Protein-protein interactions
• Protein interaction sites

Critical to most biological processes
Potential drug targets
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Analysing hMLH1-MBD4 interactions
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human MutL homolog
Methyl binding domain
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Functions of MBD4
Methyl binding domain
Binds methylated DNA
Tumor supresor
T/U glycosylase
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Functions of hMLH1
human MutL homolog
Depending on the interaction partner
Corects base-base mismatches
Recombination
Removes insertions and deletions
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Yeast two-hybrid system (Y2H)
-

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Screening protein-protein interactions
Cotransformation
Strong interactions (HIS, ADE)
Weak interactions (HIS)
Medium without Leu, Trp, His, Ade
Medium without Leu, Trp
Medium without Leu, Trp, His
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Prediction of hMLH1 protein interaction sites
Jan Kosinski
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hMLH1
human MutL homolog
NTD N terminal domain
CTD C terminal domain
756
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hMLH1 MBD4
Y2H
756

wt hMLH1
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/-
-

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/-
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hMLH1 mutations
L574P P640S P648L P648S
Jan Kosinski
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Measuring the strength of interaction
ONPG
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hMLH1 mutants-MBD4
hMLH1 mutants
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Mutations abolishing hMLH1-MBD4 interaction
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Summary
Bioinformaticians Experimentalists
Successful Protein Analysis
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www.genesilico.pl
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Acknowledgements
Phd Janusz Bujnicki Phd Krzysztof Skowronek
Elzbieta Purta Sebastian Pawlak
Katarzyna Kaminska Jan
Kosinski
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Thank you for your attention