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Cristallographie de deux enzymes : mutant de l'uridine monophosphate kinase d' E. coli insensible à la régulation allostérique et la cytokinine oxydase

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par Ahmed Meksem
Institut National Agronomique Paris Grignon - Master en Nutrition Santé 2006
  

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SUR LE WEB:

- NCBI Reference Sequence project : ftp://ftp.ncbi.nih.gov/refseq/release/

- RCSB Protein Data Bank : http://www.rcsb.org/pdb/Welcome.do/

- L'essentiel sur les cytokinines : http://www.inrp.fr/biotic/morpho/html/cytokinines.htm

Annexes

Annexe I : Crystal Screen 1

1. 0.02 M Calcium chloride dihydrate, 0.1 M Sodium acetate trihydrate pH 4.6, 30% v/v (+/-)-2-Methyl-2,4-pentanediol

2. 0.4 M Potassium sodium tartrate tetrahydrate

3. 0.4 M Ammonium phosphate monobasic

4. 0.1 M Tris hydrochloride pH 8.5, 2.0 M Ammonium sulfate

5. 0.2 M Sodium citrate tribasic dihydrate, 0.1 M HEPES sodium pH 7.5, 30% v/v (+/-)-2-Methyl-2,4-pentanediol

6. 0.2 M Magnesium chloride hexahydrate, 0.1 M Tris hydrochloride pH 8.5, 30% w/v Polyethylene glycol 4000

7. 0.1 M Sodium cacodylate pH 6.5, 1.4 M Sodium acetate trihydrate

8. 0.2 M Sodium citrate tribasic dihydrate, 0.1 M Sodium cacodylate pH 6.5, 30% v/v 2-Propanol

9. 0.2 M Ammonium acetate, 0.1 M Sodium citrate tribasic dihydrate pH 5.6, 30% w/v Polyethylene glycol 4000

10. 0.2 M Ammonium acetate, 0.1 M Sodium acetate trihydrate pH 4.6, 30% w/v Polyethylene glycol 4000

11. 0.1 M Sodium citrate tribasic dihydrate pH 5.6, 1.0 M Ammonium phosphate monobasic

12. 0.2 M Magnesium chloride hexahydrate, 0.1 M HEPES sodium pH 7.5, 30% v/v 2-Propanol

13. 0.2 M Sodium citrate tribasic dihydrate, 0.1 M Tris hydrochloride pH 8.5, 30% v/v Polyethylene glycol 400

14. 0.2 M Calcium chloride dihydrate, 0.1 M HEPES sodium pH 7.5, 28% v/v Polyethylene glycol 400

15. 0.2 M Ammonium sulfate, 0.1 M Sodium cacodylate pH 6.5, 30% w/v Polyethylene glycol 8000

16. 0.1 M HEPES sodium pH 7.5, 1.5 M Lithium sulfate monohydrate

17. 0.2 M Lithium sulfate monohydrate, 0.1 M Tris hydrochloride pH 8.5, 30% w/v Polyethylene glycol 4000

18. 0.2 M Magnesium acetate tetrahydrate, 0.1 M Sodium cacodylate pH 6.5, 20% w/v Polyethylene glycol 8000

19. 0.2 M Ammonium acetate, 0.1 M Tris hydrochloride pH 8.5, 30% v/v 2-Propanol

20. 0.2 M Ammonium sulfate, 0.1 M Sodium acetate trihydrate pH 4.6, 25% w/v Polyethylene glycol 4000

21. 0.2 M Magnesium acetate tetrahydrate, 0.1 M Sodium cacodylate pH 6.5, 30% v/v (+/-)-2-Methyl-2,4-pentanediol

22. 0.2 M Sodium acetate trihydrate, 0.1 M Tris hydrochloride pH 8.5, 30% w/v Polyethylene glycol 4000

23. 0.2 M Magnesium chloride hexahydrate, 0.1 M HEPES sodium pH 7.5, 30% v/v Polyethylene glycol 400

24. 0.2 M Calcium chloride dihydrate, 0.1 M Sodium acetate trihydrate pH 4.6, 20% v/v 2-Propanol

25. 0.1 M Imidazole pH 6.5, 1.0 M Sodium acetate trihydrate

26. 0.2 M Ammonium acetate, 0.1 M Sodium citrate tribasic dihydrate pH 5.6, 30% v/v (+/-)-2-Methyl-2,4-pentanediol

27. 0.2 M Sodium citrate tribasic dihydrate, 0.1 M HEPES sodium pH 7.5, 20% v/v 2-Propanol

28. 0.2 M Sodium acetate trihydrate, 0.1 M Sodium cacodylate pH 6.5, 30% w/v Polyethylene glycol 8000

29. 0.1 M HEPES sodium pH 7.5, 0.8 M Potassium sodium tartrate tetrahydrate

30. 0.2 M Ammonium sulfate, 30% w/v Polyethylene glycol 8000

31. 0.2 M Ammonium sulfate, 30% w/v Polyethylene glycol 4000

32. 2.0 M Ammonium sulfate

33. 4.0 M Sodium formate

34. 0.1 M Sodium acetate trihydrate pH 4.6, 2.0 M Sodium formate

35. 0.1 M HEPES sodium pH 7.5, 0.8 M Sodium phosphate monobasic monohydrate, 0.8 M Potassium phosphate

monobasic

36. 0.1 M Tris hydrochloride pH 8.5, 8% w/v Polyethylene glycol 8000

37. 0.1 M Sodium acetate trihydrate pH 4.6, 8% w/v Polyethylene glycol 4000

38. 0.1 M HEPES sodium pH 7.5, 1.4 M Sodium citrate tribasic dihydrate

39. 0.1 M HEPES sodium pH 7.5, 2% v/v Polyethylene glycol 400, 2.0 M Ammonium sulfate

40 0.1 M Sodium citrate tribasic dihydrate pH 5.6, 20% v/v 2-Propanol, 20% w/v Polyethylene glycol 4000

41. 0.1 M HEPES sodium pH 7.5, 10% v/v 2-Propanol, 20% w/v Polyethylene glycol 4000

42. 0.05 M Potassium phosphate monobasic, 20% w/v Polyethylene glycol 8000

43. 30% w/v Polyethylene glycol 1500 : Conditions de cristallisation de la CKOm

44. 0.2 M Magnesium formate dihydrate

45. 0.2 M Zinc acetate dihydrate, 0.1 M Sodium cacodylate pH 6.5, 18% w/v Polyethylene glycol 8000

46. 0.2 M Calcium acetate hydrate, 0.1 M Sodium cacodylate pH 6.5 , 18% w/v Polyethylene glycol 8000

47. 0.1 M Sodium acetate trihydrate pH 4.6, 2.0 M Ammonium sulfate

48. 0.1 M Tris hydrochloride pH 8.5, 2.0 M Ammonium phosphate monobasic

Annexe II : Crystal Screen 2

1. 10% PEG 6000, 2.0 M Sodium Chloride : Conditions de cristallisation de l'UMPKeco D93A

2. 0.5 M Sodium Chloride, 0.01 M CTAB, 0.01 M Magnesium Chloride

3. 25% Ethylene Glycol

4. 35% Dioxane

5. 5% iso-Propanol, 2.0 M Ammonium Sulfate

6. 1.0 M Imidazole pH 7.0

7. 10% PEG 1000, 10% PEG 8000

8. 10% Ethanol, 1.5 M Sodium Chloride

9. 2.0 M Sodium Chloride, 0.1 M Na Acetate pH 4.6

10. 30% MPD, 0.1 M Na Acetate pH 4.6, 0.2 M Sodium Chloride

11. 1.0 M 1,6 Hexanediol, 0.1 M Na Acetate pH 4.6, 0.01 M Cobalt Chloride

12. 30% PEG 400, 0.1 M Na Acetate pH 4.6, 0.1 M Cadmium Chloride

13. 30% PEG MME 2000, 0.1 M Na Acetate pH 4.6, 0.2 M Ammonium Sulfate

14. 2.0 M Ammonium Sulfate, 0.1 M Na Citrate pH 5.6, 0.2 M K/Na Tartrate

15. 1.0 M Lithium Sulfate, 0.1 M Na Citrate pH 5.6, 0.5 M Ammonium Sulfate

16. 2% Polyethyleneimine, 0.1 M Na Citrate pH 5.6, 0.5 M Sodium Chloride

17. 35% tert-Butanol, 0.1 M Na Citrate pH 5.6

18. 10% Jeffamine M-600, 0.1 M Na Citrate pH 5.6, 0.01 M Ferric Chloride

19. 2.5 M 1,6 Hexanediol, 0.1 M Na Citrate pH 5.6

20. 1.6 M Magnesium Sulfate, 0.1 M MES pH 6.5

21. 2.0 M Sodium Chloride, 0.1 M MES pH 6.5, 0.2 M Na/K Phosphate

22. 12% PEG 20,000, 0.1 M MES pH 6.5

23. 10% Dioxane, 0.1 M MES pH 6.5, 1.6 M Ammonium Sulfate

24. 30% Jeffamine M-600, 0.1 M MES pH 6.5, 0.05 M Cesium Chloride

25. 1.8 M Ammonium Sulfate, 0.1 M MES pH 6.5, 0.01 M Cobalt Chloride

26. 30% PEG MME 5000, 0.1 M MES pH 6.5, 0.2 M Ammonium Sulfate

27. 25% PEG MME 550, 0.1 M MES pH 6.5, 0.01 M Zinc Sulfate

28. 1.6 M Sodium Citrate pH 6.5

29. 30% MPD, 0.1 M Hepes pH 7.5, 0.5 M Ammonium Sulfate

30. 10% PEG 6000, 0.1 M Hepes pH 7.5, 5% MPD

31. 20% Jeffamine M-600, 0.1 M Hepes pH 7.5

32. 1.6 M Ammonium Sulfate, 0.1 M Hepes pH 7.5, 0.1 M Sodium Chloride

33. 2.0 M Ammonium Formate, 0.1 M Hepes pH 7.5

34. 1.0 M Sodium Acetate, 0.1 M Hepes pH 7.5, 0.05 M Cadmium Sulfate

35. 70% MPD, 0.1 M Hepes pH 7.5

36. 4.3 M Sodium Chloride, 0.1 M Hepes pH 7.5

37. 10% PEG 8000, 0.1 M Hepes pH 7.5, 8% Ethylene Glycol

38. 20% PEG 10,000, 0.1 M Hepes pH 7.5

39. 3.4 M 1,6 Hexanediol, 0.1 M Tris pH 8.5, 0.2 M Magnesium Chloride

40. 25% tert-Butanol, 0.1 M Tris pH 8.5

41. 1.0 M Lithium Sulfate, 0.1 M Tris pH 8.5, 0.01 M Nickel (II) Chloride

42. 12% Glycerol, 0.1 M Tris pH 8.5, 1.5 M Ammonium Sulfate

43. 50% MPD, 0.1 M Tris pH 8.5, 0.2 M Ammonium Phosphate

44. 20% Ethanol, 0.1 M Tris pH 8.5

45. 20% PEG MME 2000, 0.1 M Tris pH 8.5, 0.01 M Nickel (II) Chloride

46. 20% PEG MME 550, 0.1 M Bicine pH 9.0, 0.1 M Sodium Chloride

47. 2.0 M Magnesium Chloride, 0.1 M Bicine pH 9.0

48. 10% PEG 20,000, 0.1 M Bicine pH 9.0, 2% Dioxane

Résumé

La fonction biologique d'une protéine dépend étroitement de sa structure 3D. La cristallographie est la seule méthode qui permette sa détermination à résolution atomique et sans limitation de la masse moléculaire. Associée à la mutagenèse dirigée, cette technique constitue un outil clé pour la compréhension des mécanismes enzymatiques. Nous l'avons utilisée pour deux enzymes :

Uridine monophosphate kinase d'E. coli : Cette enzyme est certainement essentielle à la croissance bactérienne et n'a pas d'homologue chez l'homme, ce qui en fait une cible potentielle d'antibiotiques. Elle est hexamétrique et régulée par le GTP, un effecteur allostérique. La structure de l'enzyme avec GTP n'explique

pas simplement l'allostérie. Des cristaux du mutant D93A (non régulé par le GTP) ont été obtenus avec du GTP.

Les données de diffraction des rayons X ont été enregistrées à une résolution de 3 ?. Le jeu de données semble très difficile à exploiter.

Cytokinine oxydase du maïs : C'est une flavoenzyme monomérique à cofacteur FAD, elle clive par oxydation les cytokinines (phytohormones intervenant dans la croissance et le développement des plantes) et les rend biologiquement inactives. Plusieurs mutants de résidus proches du FAD avaient été produits par mutagenèse dirigée. Des cristaux de la CKOm sauvage (pour infiltration) et du mutant D169E (avec le substrat,

IP) ont étés obtenus, les données de diffraction ont été enregistrées à 1,8 ?. Le traitement des données du complexe CKOm sauvage-inhibiteur HA-8 (2,4 ?) et du complexe mutant D169E-substrat IP (2,0 ?) a été réalisé. Le phasage sera entrepris prochainement.

Mots clés : Protéine, Cristallographie, Uridine monophosphate kinase d'E. coli, Cytokinine oxydase de maïs, Mutagenèse dirigée.

Summary

The biological function of a protein depends on its 3D structure. Crystallography is the only method which allows its determination at atomic resolution and without limitation of the molecular weight. Associated

to site-directed mutagenesis, this technique constitutes a key tool to understand the structural basis of the enzymatic mechanisms. We applied it for two enzymes:

Uridine monophosphate kinase of E. coli: it is hexametric and is regulated by GTP, an allosteric effector. This enzyme is certainly essential to bacterial growth and does not have any counterpart in human. Crystals of mutant D93A (not controlled by the GTP) have been obtained with GTP. X-ray diffraction data have been collected to 3 ? resolution. These data are extremely difficult to process.

Cytokinine oxydase of maize: it is a monomeric flavoenzyme with FAD cofactor; it cleaves by oxidation

the cytokinines (phytohormones involved in plant growth and development) and inactivates them. Several mutants of residues close to FAD have been produced by site-directed mutagenesis. Crystals of wild-type CKOm (for infiltration) and of D169E variant (with the IP substrate) have been obtained, the diffraction data have been recorded to 1,8 ?. Data processing of CKO-HA-8 (2,4 ?) and D169E variant with IP (2,0 ?) have been successful. Phasing should be soon achieved.

Key words: Protein, Crystallography, Uridine monophosphate kinase from E. coli, Cytokinine oxydase

from maize, site-directed mutagenesis.

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