1 / 12

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

raquel
Télécharger la présentation

Kristýna Poncová, Iulia Iermak

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related