kristýna poncová, iulia iermak 28. 07. 2011 crystallization of the members of multistep signaling...
TRANSCRIPT
Kristýna Poncová, Iulia Iermak
28. 07. 2011
Crystallization of the members of multistep signaling system from Arabidopsis thaliana
SUPERVISOR: Mgr. Oksana Degtjarik
AUTHORS:
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)
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
METHODSProcess of crystallization
nucleation growth of crystals
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
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
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
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
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
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
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
Thanks for your attention!
• Acknowledgements:- to the organizers of the
Summer School- to my supervisor Mgr.
Oksana Degtjarik - to my collaborator Iulia
Iermak