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

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: Arabidopsis histidine phosphotransfer protein (AHP2) Arabidopsis histidine kinase (CKI RD ) 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

4. METHODS Process of crystallization nucleation growth of crystals

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

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

7. RESULTS AHP2 crystals c= 5 mg/ml 0,1M MES 1,6M MgSO 4 ·7H 2 O pH=6,5 mA6H crystals c=6 mg/ml 1,0 M Imidazole pH=7,0 2,0 M Ammonium Sulfate 5 % iso-propanol 1,0 M Imidazole pH=7,0 0.5 mm

8. RESULTS CKI RD 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

9. RESULTS-COMPLEXES AHP2 + CKI RD c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO 4 *7H 2 O pH=6,3 protein:precipitant 2:1 AHP2 + CKI RD c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO 4 *7H 2 O pH=5,7 protein:precipitant 2:1 AHP2 + mA6H c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO 4 *7H 2 O pH=5,7 protein:precipitant 2:1 AHP2 + mA6H c=5 mg/ml, ratio 1:1 0,1M MES 1,6M MgSO 4 *7H 2 O pH=5,7 protein:precipitant 2:2 1 mm 0,5 mm

10. How to determine if we crystallized a complex- SDS PAGE analysis 82345671910 kDa 150 100 75 50 37 25 20 1 – marker (Bio-Rad) 2 – protein AHP2 3 – antibody mA6H 4 – histidin kinase CKI RD 5 – crystal AHP2+mA6H 6 – crystal AHP2+mA6H 7 – crystal AHP2+mA6H 8 – crystal AHP2+CKI RD 9 – mother liquid from AHP2+mA6H 10 – crystal AHP2+CKI RD

11. Conclusions -We obtained 3D crystals of AHP2 and its complexes (with CKI RD and scFv mA6H) and crystals of CKI RD 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