Download - Kristýna Poncová, Iulia Iermak
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Kristýna Poncová, Iulia Iermak
28. 07. 2011
Crystallization of the members of multistep signaling system from Arabidopsis thaliana
SUPERVISOR: Mgr. Oksana Degtjarik
AUTHORS:
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The multistep signaling system
• Crucial sensing and responding mechanism in higher plants
• Modulates response for plant´s hormons cytokinins
• Hybrid receptor His kinases,
• 2 classes of response regulator proteins (A and B)
• 6 His-containing phosphotransfer proteins (AHPs)
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Crystallization trials
Proteins from Arabidopsis:
• Arabidopsis histidine phosphotransfer protein (AHP2)
• Arabidopsis histidine kinase (CKIRD)
• scFv mA6H - single chain fragment variable of antibody against AHP2
Goal to grow crystals suitable for X-ray analysis and to determine its exact structure
Model proteins
• Lysozyme
• Thaumatin
Arabidopsis thaliana
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METHODSProcess of crystallization
nucleation growth of crystals
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Hanging drop
Thaumatin crystalsc=25 mg/ml 1,5 M sodium tartratepH=6,5protein:precipitant 3:1
Sitting drop
Thaumatin crystalsc=50 mg/ml 1,5 M sodium tartratepH=6,5protein:precipitant 3:1
0.2 mm
0.2 mm
METHODS-vapour diffusion
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Microbatch under the oil
Thaumatin crystals c=15 mg/ml 1,5 M sodium tartratepH=6,5protein:precipitant 3:1
Free interface diffusion (in capillaries)
Lysozyme c= 50mg/ml NaAc pH 4.7; 12 % NaCl.
0.2 mm
0.5 mm
0.5 mm
METHODS
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RESULTS
• AHP2 crystals c= 5 mg/ml 0,1M MES 1,6M MgSO4·7H2O pH=6,5
• mA6H crystals c=6 mg/ml 1,0 M Imidazole pH=7,0
2,0 M Ammonium Sulfate5 % iso-propanol
1,0 M Imidazole pH=7,0
0.5 mm0.5 mm
0.5 mm 0.5 mm
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RESULTS
• CKIRD crystals c=13,6mg/ml
2.54M ammonium sulfate
16% glycerol
acetate buffer pH 5.05
• Optimalisation trials did not improve crystal size
0,5mm
0.2 mm
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RESULTS-COMPLEXES
AHP2 + CKIRD
c=5 mg/ml, ratio 1:10,1M MES 1,6M MgSO4*7H2O pH=6,3protein:precipitant 2:1
AHP2 + CKIRD
c=5 mg/ml, ratio 1:10,1M MES 1,6M MgSO4*7H2O pH=5,7protein:precipitant 2:1
AHP2 + mA6Hc=5 mg/ml, ratio 1:10,1M MES 1,6M MgSO4*7H2OpH=5,7protein:precipitant 2:1
AHP2 + mA6Hc=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO4*7H2OpH=5,7protein:precipitant 2:2
1 mm1 mm 0,5 mm 0,5 mm
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How to determine if we crystallized a complex-SDS PAGE analysis
82 3 4 5 6 71 9 10
kDa150100
75
50
37
25
20
1 – marker (Bio-Rad)2 – protein AHP23 – antibody mA6H4 – histidin kinase CKIRD
5 – crystal AHP2+mA6H6 – crystal AHP2+mA6H7 – crystal AHP2+mA6H8 – crystal AHP2+CKIRD
9 – mother liquid from AHP2+mA6H10 – crystal AHP2+CKIRD
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Conclusions
- We obtained 3D crystals of AHP2 and its complexes (with CKIRD and scFv mA6H) and crystals of CKIRD X-ray analysis.
- Conditions for crystallization of mH6H were screened and data will be used for further optimalization
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Thanks for your attention!
• Acknowledgements:- to the organizers of the
Summer School- to my supervisor Mgr.
Oksana Degtjarik - to my collaborator Iulia
Iermak