Detecting salt concentration dependence of the WNK1 kinase

Rachael Bergman

Eastfield Community College

Advisor Thomas Moon

Elizabeth Goldsmith Lab

June – July 2010

Experimental QuestionWhat we want to know:

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

WNK1 Kinase

Adapted from Richardson, C.; Alessi, D.; J. Cell Sci. 121(20)3293-3304Xu, B.; Min, X.; Stippec, S.; Lee, B..; Goldsmith, E.; Cobb, M.; JBC 277(50)48456-48462

Figure adopted from Thomas Moon

2382

KinaseDomain

1RFXV

194483

P

S382

Auto-InhibitoryDomain

S378

WNK1

WNK1 Inactive Structure

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

Importance of WNK1WNK1 has been linked to hypertensionWNK1 has been linked to synaptic vesicle fusionWNK1 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)3293-3304Figure adopted from Thomas Moon

Obtaining WNK1Grow mass quantities of WNK1 in E. coli

bacteriaLyse the cellsSeparate protein from cellsPurify the protein

6xHIS tag

WNK1 193-482 S382A

194 483

S382AS378

RFXV

P

KinaseDomain

Elution Volume (mL)

0 20 40 60

Abs

orba

nce

(m

AU

)

0

500

1000

1500

2000

Nickel Column WNK1 194-483 S382A

Elution Volume (mL)

20 30 40 50 60

Abs

orba

nce

(m

AU

)

0

200

400

600

800

1000

1 2 3 4 5

L 1 2 3 4 5

WNK1

Mono Q WNK1 194-483 S382A

TEV Cleavage of 6xHIS TagLa

dder

Befor

e TEV

After T

EV

Ni. Elua

nt

After T

EV

Befor

e TEV

Ladd

er

WNK1 WNK1

Elution Volume (mL)

40 45 50 55 60 65 70

Ab

sorb

an

ce (

mA

U)

0

20

40

60

80

100

120

140

160

180

200

Gel Filtration WNK1 194-483 S382A

Elution Volume (mL)

0 10 20 30 40 50

Ab

sorb

an

ce (

mA

U)

-50

0

50

100

150

200

250

Mono Q WNK1 194-483 S382A

1 2

L 1 2

WNK1

Plan1) WNK1 194-483 inactive kinase domain binds to WNK1

482-573 autoinhibitory domain by gel filtration2)Measure binding as a function of [NaCl] by gel filtration3) Problem:

Lost protein in final purification step4) Solution:

Use fluorescence to measure protein stability with NaCl and autoinhibitory domain.

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.000.00

0.20

0.40

0.60

0.80

1.00

1.20

1mM NaCl 10mM NaCl 20mM NaCl 50mM NaCl 75mM NaCl 100mM NaCl 150mM NaCl

Temperature (C)

Norm

alized F

luore

scence

[NaCl] Dependence on KD Stability

Comparative Curves for [NaCl] Dependence

ConclusionsThe 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.

AcknowledgmentsThomas MoonDr. GoldsmithGoldsmith Lab