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

Rachael Bergman

Eastfield Community College

Advisor Thomas Moon

Elizabeth Goldsmith Lab

June – July 2010


Experimental Question

  • What we want to know:

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


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

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

Figure adopted from Thomas Moon


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

Adapted from Richardson, C.; Alessi, D.; J. Cell Sci. 121(20)3293-3304

Figure adopted from Thomas Moon


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


NickelColumn WNK1 194-483 S382A


Mono Q WNK1 194-483 S382A

WNK1

L

1

2

3

4

5

1

2

3

4

5


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


Mono Q WNK1 194-483 S382A

WNK1

L

1

2

1

2


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.


[NaCl] Dependence on KD Stability


Comparative Curves for [NaCl] Dependence


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

  • Thomas Moon

  • Dr. Goldsmith

  • Goldsmith Lab


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