Detecting salt concentration dependence of the wnk1 kinase
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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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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

Experimental Question

  • What we want to know:

    • Does WNK1 194-483 S382A have a NaCl concentration dependency on kinase stability?


Wnk1 kinase

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


Wnk1 inactive structure

WNK1 Inactive Structure

Min, X.; Lee, B.; Cobb, M.; Goldsmith, E. Structure 12 1303-1311

Figure adopted from Thomas Moon


Importance of wnk1

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.


Wnk1 as a regulator of ion homeostasis

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


Obtaining wnk1

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


Nickel column wnk1 194 483 s382a

NickelColumn WNK1 194-483 S382A


Mono q wnk1 194 483 s382a

Mono Q WNK1 194-483 S382A

WNK1

L

1

2

3

4

5

1

2

3

4

5


Detecting salt concentration dependence of the wnk1 kinase

TEV Cleavage of 6xHIS Tag

WNK1

WNK1

Ladder

Ladder

After TEV

After TEV

Ni. Eluant

Before TEV

Before TEV


Gel filtration wnk1 194 483 s382a

Gel Filtration WNK1 194-483 S382A


Mono q wnk1 194 483 s382a1

Mono Q WNK1 194-483 S382A

WNK1

L

1

2

1

2


Detecting salt concentration dependence of the wnk1 kinase

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.


Detecting salt concentration dependence of the wnk1 kinase

[NaCl] Dependence on KD Stability


Comparative curves for nacl dependence

Comparative Curves for [NaCl] Dependence


Conclusions

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.


Acknowledgments

Acknowledgments

  • Thomas Moon

  • Dr. Goldsmith

  • Goldsmith Lab


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