1. Stability Analysis of Cystic
Fibrosis Transmembrane
Conductance Regulator via
Tryptic Digestion
Chris Holmes, Biophysical Society
John Riordan (PI) & Timothy Jensen (Mentor) , CF Center

2. Symptoms of Cystic Fibrosis (CF)
http://smithbiologyp3.wikispaces.com/Cystic+Fibrosis
http://upload.wikimedia.org/wikipedia/commons/4/4e/ClubbingCF.JPG
http://1.bp.blogspot.com/-VoDtcyKhUew/T0IT9WdRpvI/AAAAAAAAAE4/utY1XRNhuFE/s640/Caylee's+1st+Vest+Treatment+010edit2.jpg
Background Methods Results Conclusions

3. Cystic Fibrosis: The Resultant from the Disruption
of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR)
http://4.bp.blogspot.com/-NypSdLKYx_0/T38RrbomnwI/AAAAAAAAABw/wZpOLyNcXz0/s1600/cf-1.jpg
Background Methods Results Conclusions

4. Acquiring a Complete CFTR Crystal
Structure is Presently Unsuccessful
Molinski, S.; Eckford, P. D. W.; Pasyk, S.; Ahmadi, S.; Chin, S.; Bear, C. E. Functional rescue of F508del-CFTR using small
molecule correctors. Frontiers in Pharmacology 2012, 3.
Background Methods Results Conclusions

5. Limited Tryptic Digestion and Sonication of CFTR
Western Blot of Digested CFTR
Repeat with Mutant CFTR and Correctors
Procedure for Indirectly Determining
the Structural Stability of CFTR
Background Methods Results Conclusions

6. Expectations and Objective from the
Tryptic Digestion of CFTR
 Investigate the structural stability of CFTR
 Wild-type CFTR (natural) and ΔF508 CFTR (mutated)
 Adenosine triphosphate (ATP) and a non-hydrolysable ATP-
analogue (AMP-PNP)
 VX-809 (Lumacaftor) was used because of its success with CF
 Currently in phase three of clinical trials
http://upload.wikimedia.org/wikipedia/
commons/d/d3/Lumacaftor_skeletal.svg
http://upload.wikimedia.org/wikipedia/
commons/0/07/ATP_structure.svg
http://patentimages.storage.googleapis.com/WO2003076333A2/imgf000010_0002.png
Background Methods Results Conclusions

7. Using Tryptic Digestion: Cleavage
Locations at Lysine Residues
Background Methods Results Conclusions
Henderson, M. J.; Singh, O. V.; Zeitlin, P. L. Applications of proteomic technologies for understanding the premature
proteolysis of CFTR. Expert Rev Proteomics 2010, 7, 473–486.
 Trypsin cleaves the protein on the carboxyl side of lysine or arginine
residues
 The more digestion that occurs, the more exposed those lysine and
arginine residues are.
0μL
1μL
3μL
9μL

8. Using Sonication to Permit the Passage of
Treatments and Trypsin into the CFTR Cells
Background Methods Results Conclusions
= CFTR Membrane
= Treatment (VX-809, ATP, AMP-PNP)
= Trypsin

9. Using Western Blot Band Strength as an
Indicator of Structural Stability
Background Methods Results Conclusions
The More Intense the Band
The More Intact the CFTR
The Higher the Stability

10. IR Detection Using Primary and
Secondary Antibodies
Membrane
Antigen
1° Antibody
2° Antibody
Fluorophore
Background Methods Results Conclusions
1° Antibodies = 660 & 769
2° Antibodies = IgG1 & IgG2b

11. Limited Tryptic Digestion as an
Indicator of CFTR Stability
Cui, L.; Aleksandrov, L.; Chang, X.; Hou, Y.; He, L.; Hegedus, T.; Gentzsch, M.; Aleksandrov, A.; Balch, W. E.;
Riordan, J. R. Domain Interdependence in the Biosynthetic Assembly of CFTR. J. Mol. Biol. 2007, 365, 981-994
Background Methods Results Conclusions

12. NBD 2
Western Blotting of Wild-Type CFTR
after Tryptic Digestion
NBD 1
AMP-PNP Wild-Type ATP VX-809
0µL 1µL 3µL 9µL
Background Methods Results Conclusions
~170 kDa

13. Data Quantification of Wild-Type CFTR
Membranes is Significant
Background Methods Results Conclusions
0
10
20
30
40
50
60
70
80
90
100
110
0 1 3 9
NormalizedValueofInitialBand(%)
Trypsin Concentration (µL)
NBD 1
AMP-PNP
VX-809
WT
ATP
0
10
20
30
40
50
60
70
80
90
100
110
0 1 3 9
NormalizedValueofInitialBand(%)
Trypsin Concentration (µL)
NBD 2
AMP-PNP
VX-809
WT
ATP
*
*
*
*

14. NBD 2
Western Blotting of ΔF508 CFTR
after Tryptic Digestion
NBD 1
AMP-PNP ΔF508 ATP VX-809
0µL1µL3µL 9µL
Background Methods Results Conclusions

15. Data Quantification of ΔF508 CFTR
Membranes is NOT Significant
Background Methods Results Conclusions
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 1 3 9
NormalizedValueofInitialBand(%)
Trypsin Concentration (µL)
NBD 1
AMP-PNP
VX-809
ΔF
ATP
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 1 3 9
NormalizedValueofInitialBand(%)
Trypsin Concentration (µL)
NBD 2
AMP-PNP
VX-809
ΔF
ATP
At least not yet!
<1%
100100

16. Adjusting the Quantified Band from the Full-
Length CFTR to Approximately Half-Length
CFTR for a More Definitive Reading
170 kDa
Background Methods Results Conclusions
AMP-PNP ΔF508 ATP VX-809
0µL 1µL 3µL 9µLMWM 0µL 1µL 3µL 9µLMWM 0µL 1µL 3µL 9µLMWM 0µL 1µL 3µL 9µLMWM
~72 kDa

17. The Difference Between 170 kDa and
72 kDa in CFTR Membranes
Cui, L.; Aleksandrov, L.; Chang, X.; Hou, Y.; He, L.; Hegedus, T.; Gentzsch, M.; Aleksandrov, A.; Balch, W. E.;
Riordan, J. R. Domain Interdependence in the Biosynthetic Assembly of CFTR. J. Mol. Biol. 2007, 365, 981-994
Background Methods Results Conclusions

18. 0
2
4
6
8
10
12
14
16
18
0 1 3 9
NormalizedValueofInitialBand(%)
Trypsin Concentration (µL)
NBD 1
AMP-PNP
VX-809
ΔF
ATP
Data Quantification of ΔF508 CFTR
Membranes IS Significant
Background Methods Results Conclusions
>1%
0
2
4
6
8
10
12
14
16
18
0 1 3 9
NormalizedValueofInitialBand(%)
Trypsin Concentration (µL)
NBD 2
AMP-PNP
VX-809
ΔF
ATP
* *
*
100 100

19. Implications of the Stabilizing Effects of
ATP and AMP-PNP
Background Methods Results Conclusions
Bound ATP and AMP-PNP
Favorable Interactions and Conformational
Adjustments
Lower Susceptibility to Proteolysis
(Tryptic Digestion) & Increased Stability

20. Future Directions for the Stability
Analysis of CFTR
Background Methods Results Conclusions
 Since VX-809 was not particularly effective
 Investigate the effects of VX-661 and/or VX-770
 Since AMP-PNP and ATP were effective
 Investigate other ATP analogues or treatments that would
extort the stabilizing effects of the ATP binding pocket
 Since the ΔF508 mutation only represents one class of CFTR
mutations
 Investigate the stabilities of other mutations(e.g. G551D)
 Since the experiment was small scale and did not account
for other mechanisms
 Investigate the treatments in vivo

21. Acknowledgments
Special thanks to the University of Mount Union, the Department of Chemistry
and Biochemistry, UNC, the Biophysical Society, and the Department of
Biochemistry and Biophysics for providing me with this opportunity
University of North Carolina
In the Laboratory:
 Dr. John Riordan
 Dr. Tim Jensen
 Dr. Luba Aleksandrov
 Mr. Mohamed Dumbuya
In the Summer Program:
 Dr. Mike Jarstfer
 Dr. Barry Lentz
 Lisa Phillippie
 Ellen Mackall
 Dr. Jaime Campbell-Fox
 Patrick McCarter
 Lior Vered
University of Mount Union
Mentoring:
 Dr. Robert Woodward
 Dr. Keith Miller
Student Assistants:
 Amanda Dragan

22. Questions?
http://www.waid-observatory.com/arp194-2012-08-10-HLA-1092.html