Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors: = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Würzburg
2015
|
| Subjects: | |
| Online Access: | Volltext Inhaltsverzeichnis |
| Physical Description: | VII, 135 Blatt Illustrationen |
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MARC
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| 100 | 1 | |a Stumpf, Anette D. |d 1985- |e Verfasser |0 (DE-588)1096198851 |4 aut | |
| 245 | 1 | 0 | |a Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors |b = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |c submitted by Anette D. Stumpf from Aalen |
| 246 | 1 | 1 | |a Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |
| 264 | 1 | |a Würzburg |c 2015 | |
| 300 | |a VII, 135 Blatt |b Illustrationen | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2015 | ||
| 546 | |a Zusammenfassung in deutscher und englischer Sprache | ||
| 650 | 0 | 7 | |a Adenosinrezeptor |0 (DE-588)4327002-5 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a G-Protein gekoppelter Rezeptor |0 (DE-588)7723480-7 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Fluoreszenz-Resonanz-Energie-Transfer |0 (DE-588)4773018-3 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Adenosinrezeptor |0 (DE-588)4327002-5 |D s |
| 689 | 0 | 1 | |a Fluoreszenz-Resonanz-Energie-Transfer |0 (DE-588)4773018-3 |D s |
| 689 | 0 | 2 | |a G-Protein gekoppelter Rezeptor |0 (DE-588)7723480-7 |D s |
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Record in the Search Index
| _version_ | 1804176143395323904 |
|---|---|
| adam_text | D EV ELO P M EN T O F FLU ORESCEN T F R E T RECEP TO R
SEN SORS FOR IN V E STIG A TIO N O F CO N FO RM A TIO N A L
CH AN GES IN A D EN O SIN E
A L
AND RECEP TO RS
E N TW ICK LU N G FLU ORESZEN TER F R E T
R E Z E P TO R S E N S O R E N ZUR U N TERSU CH U N G VON
K ON FO RM A TIO N SAE N D ERU N G EN IN A D EN O SIN
A L
UND
A 2
A
R E Z E P TO R E N
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG
SECTION BIOMEDICINE
SUBMITTED BY
ANETTE D. STUMPS
FROM AALEN
WUERZBURG 2015
CONTENTS
1 INTRODUCTION 1
1.1 G PROTEIN-COUPLED RE C E P TO RS
.................................................................... 1
1.1.1 CLASSIFICATION OF G P C R S
.............................................................. 1
1.1.2 GPCRS AS TARGETS FOR DRUGS - INCREASING STRUCTURAL KNOWLEDGE 2
1.1.3 ADENOSINE
RECEPTORS.......................................................................
2
1.2 RECEPTOR LIGAND IN TE RAC TIO N
S.................................................................... 8
1.2.1 DYNAMIC RECEPTOR MOVEMENT UPON LIGAND BINDING 8
1.2.2 REQUIREMENTS FOR EFFECTIVE DRUGS AT RECEPTORS. 10
1.3 FLUORESCENCE, DESCRIPTION OF FLUOROPHORES USED AND FLUORESCENCE -
BASED
TECHNIQUES...................................................................................................
11
1.3.1 FLUORESCENT PROTEINS DEVELOPED FROM GFP AND OTHER FLUORES-
CENT MOLECULES FOR PROTEIN
LABELING................................................ 11
1.3.2 THE PRINCIPLE OF SNAP AND C L I P
................................................ 12
1.3.3 FLUORESCENCE AND FLUORESCENCE RESONANCE ENERGY TRANSFER . 13
1.4 FLUORESCENCE BASED APPROACHES TO MONITOR RECEPTOR DYNAMICS AND RE-
CEPTOR LIGAND
INTERACTIONS..........................................................................
16
2 MOTIVATION 19
3 MATERIA, AND METHODS 20
3.1 M A TE RIA
L..........................................................................................................
20
3.1.1 D N A
.................................................................................................20
3.1.2 D N A
....................................................................................................20
3.1.3 CELL
LINES..............................................................................................21
3.1.4 FLUORESCENT PROTEINS AND D Y E S
........................................................
22
3.1.5 E NZYM
ES..............................................................................................22
3.1.6 B A C TE RIA
..............................................................................................22
3.1.7 PLASM IDS
.............................................................................................
22
3.1.8 C HEM
ICALS................................................................................
23
3.1.9 REAGENTS FOR CELL C U LT U R E
.......................................................
25
3.1.10 K I T S
................................................................................................26
3.1.11 BUFFERS AND REAGENT MIXES FOR ASSAYS
........................................
26
3.1.12 EXPENDABLE MATERIALS AND OTHER SUBJECTS USED
......... 27
3.1.13 M ICROSCOPES
...................................................................................
27
3.1.14 SOFTW
ARE..........................................................................................27
3.2 M E TH O D S
..............................................................................................
28
3.2.1 CELL C U LT U R E
...................................................................................
28
3.2.2 LABELING OF LIVING C E L L S
.................................................................31
3.2.3 FRET MEASUREMENT
.................................................................... 33
3.2.4 CONFOCAL M
ICROSCOPY....................................................................
35
3.2.5 GENERATION OF COMPETENT B A C TE RIA
..............................................
35
3.2.6 PURIFICATION
*
AMPLIFICATION AND PROCESSING OF PLASMID DNA . . 36
3.2.7 INHIBITORY ADENYLATE CYCLASE A S S A Y
..................................................
40
3.2.8 MEMBRANE P REP A RATIO N
.....................................................................42
3.2.9 DETERMINATION OF PROTEIN CONCENTRATION WITH HELP OF BRADFORD
A SSA Y
....................................................................................................42
3.2.10 RADIOLIGAND SATURATION BINDING A S S A Y
............................................
43
3.2.11 DATA A N A LY S IS
....................................................................................
43
4 RESULTS 45
4.1 DEVELOPMENT OF AL F13 CFP AND COMPARISON TO
A 2
A
F13 CFP .... 45
4.1.1 GENERATION OF AL F13CFP RECEPTOR S E N S O R
...................................45
4.1.2 CHARACTERIZATION OF AL F13 CFP RECEPTOR SENSOR
........ 47
4.1.3 FRET EXPERIMENTS AT AL F13 CFP AND
A 2
A
F13 CFP - COM-
PARISON REGARDING SIGNAL AMPLITUDES AND KINETICS
. . . . . . . *
48
4.1.4 DEVELOPMENT OF AL F13 CFP RECEPTOR SENSOR MUTANTS ..... 53
4.1.5 CONFOCAL ANALYSIS OF AL F13 CFP AND ITS MUTANTS ....... 56
4.1.6 FRET MEASUREMENTS OF AL F13 CFP AND ITS MUTANTS ..... 59
4.2 INVESTIGATION OF N-TERMINALLY SNAP AND CLIP TAGGED A! AND *2*
RECEPTOR SEN SO
RS..............................................................................................75
4.2.1 CHARACTERIZATION OF FLAG SNAP AIR AND FLAG SNAP A2
A
R . . . 78
4.2.2 SNAP-TAGGED RECEPTOR SENSORS - BINDING EXPERIMENTS WITH FLU-
ORESCENT ADENOSINE RECEPTOR LIG A N D S
...............................................
80
4.3 SNAP AL F13 CFP AND SNAP A% F13 C F P
........................................
88
4.3.1 DEVELOPMENT OF SNAP AL F13 CFP AND SNAP **/* F13 CFP . 89
4.3.2 CONFOCAL ANALYSIS OF SNAP AL F13 CFP AND SNAP
K 2A
F13
CFP SENSORS AND BEHAVIOR IN FRET EXPERIMENTS ........ 90
5 DISCUSSION 92
5.1 COMPARISON OF AL F13 CFP AND
*
2
A
F13 CFP REVEALED DIFFERENCES
BETWEEN THE TWO ADENOSINE RECEPTOR
SUBTYPES..............................................92
5.2 AL F13 CFP AND ITS MUTANTS REVEALED NEW INSIGHTS INTO RECEPTOR DY-
NAMICAL B E H A V IO
R...........................................................................................94
5.2.1 INVOLVEMENT OF SINGLE AMINO ACIDS IN THE AIR IN THE CELLULAR
D ISTRIB U TIO N
.......................................................................................
95
5.2.2 INVESTIGATION OF LIGAND BINDING BEHAVIOR AT THE AIR VIA FRET
M EASUREM
ENTS....................................................................................
96
5.3 COMBINING FLUORESCENCE-BASED RECEPTOR LABELING TECHNIQUES - FUTURE
D IRE C TIO N S
.....................................................................................................
101
6 SUMMARY 103
6.1 SUMMARY
.....................................................................................................
103
6.2
ZUSAMMENFASSUNG.........................................................................................104
7 ABBREVIATIONS 107
8 L*GANDS- STRUCTURES AND PROPERTIES 110
9 BIBLIOGRAPHY 113
10 PUBLICATIONS AND CONFERENCES ATTENDED 129
10.1
PUBLICATIONS..................................................................................................
129
10.2 CONFERENCES A TTE N D E D
..................................................................................
130
11 ACKNOWLEDGMENT 132
12 AFFIDAVIT/EIDESSTATTLICHE ERKLAERUNG 133
12.1 A
FFIDAVIT........................................................................................................
133
12.2 EIDESSTATTLICHE E RKLAERUNG
............................................................................
133
13 CURRICULUM VITAE
135
|
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| author | Stumpf, Anette D. 1985- |
| author_GND | (DE-588)1096198851 |
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| dewey-hundreds | 600 - Technology (Applied sciences) |
| dewey-ones | 610 - Medicine and health |
| dewey-raw | 610 |
| dewey-search | 610 |
| dewey-sort | 3610 |
| dewey-tens | 610 - Medicine and health |
| discipline | Medizin |
| format | Thesis Book |
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| indexdate | 2024-07-10T07:27:30Z |
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| language | English |
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| physical | VII, 135 Blatt Illustrationen |
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| spelling | Stumpf, Anette D. 1985- Verfasser (DE-588)1096198851 aut Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren submitted by Anette D. Stumpf from Aalen Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren Würzburg 2015 VII, 135 Blatt Illustrationen txt rdacontent n rdamedia nc rdacarrier Dissertation Julius-Maximilians-Universität Würzburg 2015 Zusammenfassung in deutscher und englischer Sprache Adenosinrezeptor (DE-588)4327002-5 gnd rswk-swf G-Protein gekoppelter Rezeptor (DE-588)7723480-7 gnd rswk-swf Fluoreszenz-Resonanz-Energie-Transfer (DE-588)4773018-3 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Adenosinrezeptor (DE-588)4327002-5 s Fluoreszenz-Resonanz-Energie-Transfer (DE-588)4773018-3 s G-Protein gekoppelter Rezeptor (DE-588)7723480-7 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-125469 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-125469 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028921625&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Stumpf, Anette D. 1985- Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren Adenosinrezeptor (DE-588)4327002-5 gnd G-Protein gekoppelter Rezeptor (DE-588)7723480-7 gnd Fluoreszenz-Resonanz-Energie-Transfer (DE-588)4773018-3 gnd |
| subject_GND | (DE-588)4327002-5 (DE-588)7723480-7 (DE-588)4773018-3 (DE-588)4113937-9 |
| title | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |
| title_alt | Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |
| title_auth | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |
| title_exact_search | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |
| title_full | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren submitted by Anette D. Stumpf from Aalen |
| title_fullStr | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren submitted by Anette D. Stumpf from Aalen |
| title_full_unstemmed | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren submitted by Anette D. Stumpf from Aalen |
| title_short | Development of fluorescent FRET receptor sensors for investigation of conformational changes in adenosine A1 and A2A receptors |
| title_sort | development of fluorescent fret receptor sensors for investigation of conformational changes in adenosine a1 and a2a receptors entwicklung fluoreszenter fret rezeptor sensoren zur untersuchung von konformationsanderungen in adenosin a1 und a2a rezeptoren |
| title_sub | = Entwicklung fluoreszenter FRET Rezeptor-Sensoren zur Untersuchung von Konformationsänderungen in Adenosin A1 und A2A Rezeptoren |
| topic | Adenosinrezeptor (DE-588)4327002-5 gnd G-Protein gekoppelter Rezeptor (DE-588)7723480-7 gnd Fluoreszenz-Resonanz-Energie-Transfer (DE-588)4773018-3 gnd |
| topic_facet | Adenosinrezeptor G-Protein gekoppelter Rezeptor Fluoreszenz-Resonanz-Energie-Transfer Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-125469 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028921625&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT stumpfanetted developmentoffluorescentfretreceptorsensorsforinvestigationofconformationalchangesinadenosinea1anda2areceptorsentwicklungfluoreszenterfretrezeptorsensorenzuruntersuchungvonkonformationsanderungeninadenosina1unda2arezeptoren AT stumpfanetted entwicklungfluoreszenterfretrezeptorsensorenzuruntersuchungvonkonformationsanderungeninadenosina1unda2arezeptoren |