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Kristýna Poncová, Iulia Iermak

28 . 07. 2011. SUPERVISOR: Mgr. Oksana Degtjarik. AUTHORS :. Kristýna Poncová, Iulia Iermak. Crystallization of the members of multistep signaling system from Arabidopsis thaliana. The multistep signaling system. Crucial sensing and responding mechanism in higher plants

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Kristýna Poncová, Iulia Iermak

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  1. 28. 07. 2011 SUPERVISOR: Mgr. Oksana Degtjarik AUTHORS: Kristýna Poncová, Iulia Iermak Crystallization of the members of multistep signaling system from Arabidopsis thaliana

  2. 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)

  3. Crystallization trials Proteins from Arabidopsis: Model proteins Lysozyme Thaumatin • Arabidopsishistidine phosphotransferprotein (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 Arabidopsis thaliana

  4. METHODSProcess of crystallization nucleation growth of crystals

  5. Hanging drop METHODS-vapour diffusion Sitting drop 0.2 mm 0.2 mm Thaumatin crystals c=50 mg/ml 1,5 M sodium tartrate pH=6,5 protein:precipitant 3:1 Thaumatin crystals c=25 mg/ml 1,5 M sodium tartrate pH=6,5 protein:precipitant 3:1

  6. Microbatch under the oil METHODS Free interface diffusion(in capillaries) 0.5 mm 0.2 mm 0.5 mm Thaumatin crystals c=15 mg/ml 1,5 M sodium tartrate pH=6,5 protein:precipitant 3:1 Lysozyme c= 50mg/ml NaAc pH 4.7; 12 % NaCl.

  7. RESULTS • AHP2 crystals • c= 5 mg/ml • 0,1M MES 1,6M MgSO4·7H2O • pH=6,5 0.5 mm 0.5 mm • mA6H crystals • c=6 mg/ml • 1,0 M Imidazole • pH=7,0 0.5 mm 0.5 mm 1,0 M Imidazole pH=7,0 2,0 M Ammonium Sulfate 5 % iso-propanol

  8. RESULTS • CKIRDcrystals c=13,6mg/ml 2.54M ammoniumsulfate 16% glycerol acetate buffer pH 5.05 • Optimalisation trials did not improve crystal size 0.2 mm 0,5mm

  9. RESULTS-COMPLEXES 1 mm 1 mm 0,5 mm 0,5 mm AHP2 + mA6H c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO4*7H2O pH=5,7 protein:precipitant 2:1 AHP2 + mA6H c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO4*7H2O pH=5,7 protein:precipitant 2:2 AHP2 + CKIRD c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO4*7H2O pH=6,3 protein:precipitant 2:1 AHP2 + CKIRD c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO4*7H2O pH=5,7 protein:precipitant 2:1

  10. How to determine if we crystallized a complex-SDS PAGE analysis kDa 150 1 – marker (Bio-Rad) 2 – protein AHP2 3 – antibody mA6H 4 – histidin kinase CKIRD 5 – crystal AHP2+mA6H 6 – crystal AHP2+mA6H 7 – crystal AHP2+mA6H 8 – crystal AHP2+CKIRD 9 – mother liquid from AHP2+mA6H 10 – crystal AHP2+CKIRD 100 75 50 37 25 20 1 2 3 4 5 6 7 8 9 10

  11. Conclusions • We obtained 3D crystals of AHP2 and its complexes(with CKIRDand scFv mA6H) and crystals of CKIRD X-ray analysis. • Conditions for crystallization of mH6H were screened and data will be used for further optimalization

  12. Thanks for your attention! • Acknowledgements: • to the organizers of the Summer School • to my supervisor Mgr. Oksana Degtjarik • to my collaborator Iulia Iermak

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