Table Of ContentDissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
A quantitative FRET approach to characterize
protein-protein interactions in living cells
Elisabeth Susanna Butz
aus
Worms, Deutschland
2017
Erklärung
Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November
2011 von Herrn Prof. Dr. Christian Wahl-Schott betreut.
Eidesstattliche Versicherung
Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet.
München, den 21.09.2017
_____________________________
(Elisabeth S. Butz)
Dissertation eingereicht am 21.09.2017
1. Gutachter: Prof. Dr. Christian Wahl-Schott
2. Gutachter: Prof. Dr. Martin Biel
Mündliche Prüfung am 20.11.2017
meinen Eltern
Reid, Robert, Artist, photographer by Highsmith, Carol M.
[Second Floor, North Corridor.
Mural depicting Understanding by Robert Reid.
Library of Congress Thomas Jefferson Building, Washington D.C.].
Photograph. Retrieved from the Library of Congress,
<https://www.loc.gov.item/2007684512/>.
Wisdom is the principal thing;
therefore get wisdom; and with all thy getting, get understanding.
Proverbs 4:7
Table of contents
1 Abbreviations and symbols I
2 List of manuscripts IV
3 Summary VI
Zusammenfassung VIII
4 Introduction 1
4. 1 Principles of SE-FRET and E-FRET 1
4. 2 Calculation of binding curves 9
4. 3 Application of FRET to identify intermolecular interaction partners 13
4. 3. 1 An epilepsy-causing mutation in Kv7.2 affects calmodulin binding 15
4. 3. 2 Two-pore channel 2 and its role in the endolysosomal system 17
4. 3. 3 Peripherin 2 and its role in photoreceptor outer segments 22
5 Aims of the thesis 26
6 Short summary of manuscripts 27
6. 1 Quantifying macromolecular interactions in living cells using FRET two-
hybrid assays 27
6. 2 Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly
destabilizing epileptogenic mutation 28
6. 3 High susceptibility to fatty liver disease in two-pore channel 2-deficient mice 29
6. 4 TPC2 polymorphisms associated with a hair pigmentation phenotype in
humans result in gain of channel function by independent mechanisms 30
6. 5 Peripherin-2 couples rhodopsin to the CNG channel in outer segments of rod
photoreceptors 31
6. 6 Peripherin-2 differentially interacts with cone opsins in outer segments of cone
photoreceptors 32
6. 7 AAV Vectors for FRET-based analysis of protein-protein interactions in
photoreceptor outer segments 33
6. 8 Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting
of retinitis pigmentosa-linked peripherin-2 mutants 34
7 References 35
8 Publications 42
9 Acknowledgement 45
10 Appendix 46
Abbreviations and symbols | I
1 Abbreviations and symbols
Abbreviations
2+
Ca calcium
CaM calmodulin
CFP cyan fluorescent protein
CNG cyclic nucleotide-gated channel
CNGB1 cyclic nucleotide-gated channel subunit B1
EGF epidermal growth factor
EGFR epidermal growth factor receptor
Em emission
ES endolysosomal system
Ex excitation
FRET Förster resonance energy transfer
GFP green fluorescent protein
HEK human embryonic kidney
Kv7.2 voltage-gated potassium channel 7.2
L-opsin long wavelength opsin
LDL low density lipoprotein
mTOR mechanistic target of rapamycin
mTORC1 mechanistic target of rapamycin complex 1
NAADP nicotinic acid adenine dinucleotide phosphate
NAFLD non alcoholic fatty liver disease
nm nano meter
OS outer segment
PDGFR β platelet-derived growth factor receptor β
PIP2 phosphatidylinositol 4,5-bisphosphate
PI(3,5)P2 phosphatidylinositol 3,5-bisphosphate
rAAV recombinant adeno-associated virus
SE sensitized emission
S-opsin short wavelength opsin
SNARE soluble N-ethylmaleimide-sensitive factor attachment receptor
Stx syntaxin
Abbreviations and symbols | II
TEM transmission electron microscopy
TM transmembrane domain
TPC two-pore channel
TRP transient receptor potential
TRPML mucolipin family of TRP channels
YFP yellow fluorescent protein
Symbols
𝜀(436) molar extinction coefficient of CFP at 436 nm
𝜀
(436) molar extinction coefficient of YFP at 436 nm
Ab fraction of acceptor molecules bound by donor
Afree fraction of unbound acceptor molecules
CFPEST estimated number of CFP molecules
CFPFRET (436, 535, direct) fluorescence signal from a CFP molecule acquired through the
FRET cube
Db fraction of donor molecules bound by acceptor
Dfree fraction of unbound donor molecules
E true or efficient FRET efficiency
EA apparent FRET efficiency as determined by SE-FRET
ED apparent FRET efficiency as determined by E-FRET
EMAX maximal FRET efficiency
FC measure of sensitized emission
G calibration constant; ratio between sensitized emission and
donor dequenching
Kd,EFF dissociation constant
MA proportionality constant relating to the acceptor pathway
MD proportionality constant relating to the donor pathway
RA1 constant relating to the spectral properties of YFP
RD1 constant relating to the spectral properties of CFP
SCFP (D, 436, 480) fluorescence measurement from a donor-only sample acquired
through the CFP cube
SCFP (DA, 436, 480) fluorescence measurement from a sample expressing both
donor and acceptor acquired through the CFP cube
Abbreviations and symbols | III
SFRET (A, 436, 535) fluorescence measurement from an acceptor-only sample
acquired through the FRET cube
SFRET (D, 436, 535) fluorescence measurement from a donor-only sample acquired
through the FRET cube
SFRET (DA, 436, 535) fluorescence measurement from a sample expressing both
donor and acceptor acquired through the FRET cube
SYFP (A, 500, 535) fluorescence measurement from an acceptor-only sample
acquired through the YFP cube
SYFP (DA, 500, 535) fluorescence measurement from a sample expressing both
donor and acceptor acquired through the YFPc ube
YFPEST estimated number of YFP molecules
YFPFRET (436, 535, direct) fluorescence signal from a YFP molecule acquired through the
FRET cube
List of Manuscripts | IV
2 List of manuscripts
This thesis is based on the following publications, which are referred to in the text by their
roman numerals (I-VIII):
I. Elisabeth S. Butz*, Manu Ben-Johny*, Michael Shen, Philemon S. Yang, Lingjie
Sang, Martin Biel, David T. Yue, Christian Wahl-Schott
Quantifying macromolecular interactions in living cells using FRET two-hybrid
assays
2016, Nature Protocols, 11(12): 2470-2498
* equal contribution
II. Araitz Alberdi*, Carolina Gomis-Perez*, Ganeko Bernardo-Seisdedos, Alessandro
Alaimo, Covadonga Malo, Juncal Aldaregia, Carlos Lopez-Robles, Pilar Areso,
Elisabeth S. Butz, Christian Wahl-Schott, Alvaro Villarroel
Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly
destabilizing epileptogenic mutation
2015, Journal of Cell Science, 128(21), 4014-4023
* equal contribution
III. Christian Grimm, Lesca M. Holdt*, Cheng-Chang Chen*, Sami Hassan*, Christoph
Müller, Simone Jörs, Hartmut Cuny, Sandra Kissing, Bernd Schröder, Elisabeth S.
Butz, Bernd Northoff, Jan Castonguay, Christian A. Luber, Markus Moser, Saskia
Spahn, Renate Lüllmann-Rauch, Christina Fendel, Norbert Klugbauer, Oliver
Griesbeck, Albert Haas, Matthias Mann, Franz Bracher, Daniel Teupser, Paul Saftig,
Martin Biel, Christian Wahl-Schott
High susceptibility to fatty liver disease in two-pore channel 2-deficient mice
2014, Nature Communications, 5:4699
* equal contribution
