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: - PowerPoint PPT PresentationTRANSCRIPT
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