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