1. Stability Analysis of Cystic
Fibrosis Transmembrane
Conductance Regulator via
Tryptic Digestion
Chris Holmes, Biophysical Society
John Riordan (PI) & Timothy Jensen (Mentor) , CF Center
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
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
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
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
Digital clubbing. Airway Clearance Technique. 1500+ mutations.
Osmolarity Imbalance
(A) Full-length homology model of CFTR MSD1, blue; MSD2, yellow; NBD1, cyan; NBD2, orange; R domain, green; F508, red; (B) position of F508 at the ICL4:NBD1 interface; (C)crystal structure of NBD1 ATP, pink.
Stored at -80C to prevent autolysis (self-digestion).
Sonication was used to permit infiltration of the trypsin and treatments evenly throughout CFTR
Explain CB
Explain nbd ABC too
Embolden SD
Conclusions starting here
Explain AMP again
