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Detecting salt concentration dependence of the WNK1 kinase

Detecting salt concentration dependence of the WNK1 kinase. Rachael Bergman Eastfield Community College Advisor Thomas Moon Elizabeth Goldsmith Lab June – July 2010. Experimental Question. What we want to know:

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Detecting salt concentration dependence of the WNK1 kinase

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  1. Detecting salt concentration dependence of the WNK1 kinase Rachael Bergman Eastfield Community College Advisor Thomas Moon Elizabeth Goldsmith Lab June – July 2010

  2. Experimental Question • What we want to know: • Does WNK1 194-483 S382A have a NaCl concentration dependency on kinase stability?

  3. WNK1 Kinase S378 S382 P Kinase Domain WNK1 1 Auto-Inhibitory Domain RFXV 2382 194 483 Adapted from Richardson, C.; Alessi, D.; J. Cell Sci. 121(20)3293-3304 Xu, B.; Min, X.; Stippec, S.; Lee, B..; Goldsmith, E.; Cobb, M.; JBC 277(50)48456-48462 Figure adopted from Thomas Moon

  4. WNK1 Inactive Structure Min, X.; Lee, B.; Cobb, M.; Goldsmith, E. Structure 12 1303-1311 Figure adopted from Thomas Moon

  5. Importance of WNK1 • WNK1 has been linked to hypertension • WNK1 has been linked to synaptic vesicle fusion • WNK1 has been linked to mitotic spindle formation in mitosis.

  6. WNK1 as a Regulator of Ion Homeostasis Adapted from Richardson, C.; Alessi, D.; J. Cell Sci. 121(20)3293-3304 Figure adopted from Thomas Moon

  7. Obtaining WNK1 • Grow mass quantities of WNK1 in E. coli bacteria • Lyse the cells • Separate protein from cells • Purify the protein S378 S382A WNK1 193-482 S382A P Kinase Domain 6xHIS tag RFXV 194 483

  8. NickelColumn WNK1 194-483 S382A

  9. Mono Q WNK1 194-483 S382A WNK1 L 1 2 3 4 5 1 2 3 4 5

  10. TEV Cleavage of 6xHIS Tag WNK1 WNK1 Ladder Ladder After TEV After TEV Ni. Eluant Before TEV Before TEV

  11. Gel Filtration WNK1 194-483 S382A

  12. Mono Q WNK1 194-483 S382A WNK1 L 1 2 1 2

  13. Plan • 1) WNK1 194-483 inactive kinase domain binds to WNK1 482-573 autoinhibitory domain by gel filtration • 2)Measure binding as a function of [NaCl] by gel filtration • 3) Problem: • Lost protein in final purification step • 4) Solution: • Use fluorescence to measure protein stability with NaCl and autoinhibitory domain.

  14. [NaCl] Dependence on KD Stability

  15. Comparative Curves for [NaCl] Dependence

  16. Conclusions • The data presented was unable to support or reject the original hypothesis • The results did not show any effect on unfolding relative to the amount of WNK1 autoinhibitory domain added. • This data is inconclusive. • We were unable to accurately measure a binding constant of the autoinhibitory domain to the kinase domain. • The data shows a linear dependence on stabilization of WNK1 kinase domain and the amount of salt present in the buffer and this was expected to be a logarithmic function. • Since there is not enough data to support or refute this, the answer to our hypothesis cannot be determined.

  17. Acknowledgments • Thomas Moon • Dr. Goldsmith • Goldsmith Lab

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