1 / 17

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:

adeola
Download Presentation

Detecting salt concentration dependence of the WNK1 kinase

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related