Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs: modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields
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| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Berlin
2016
|
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | IV, 167 Seiten Illustrationen, Diagramme |
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| 100 | 1 | |a Völler, Jan-Stefan |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs |b modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields |c by Jan-Stefan Völler |
| 264 | 1 | |a Berlin |c 2016 | |
| 300 | |a IV, 167 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Freie Universität Berlin |d 2016 | ||
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Record in the Search Index
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| adam_text | TABLE OF CONTENTS
SUMMARY........................................................................................................................................1
ZUSAMMENFASSUNG.......................................................................................................................
3
1 P A R
TI................................................................................................................................................
5
1.1
INTRODUCTION............................................................................................................................7
1.1.1 ALIPHATIC FLUORINATED AMINO ACIDS IN PEPTIDE AND PROTEIN D ESIG N
..............................
7
1.1.2 SYNTHESIS O F FLUORINATED PEPTIDES AND
PROTEINS.............................................................9
1.1.3 THE GENETIC CODE IN
PROKARYOTES....................................................................................
9
1.1.4 RIBOSOMAL
TRANSLATION...................................................................................................
12
1.1.5 AMINOACYL-TRNA SYNTHETASES (AARSS)
.....................................................................
13
1.1.6 ENGINEERING THE GENETIC
CODE........................................................................................19
1.1.7 THE MODEL PEPTIDE VW 18
............................................................................................
23
1.2 AIM O F THIS STU D
Y.................................................................................................................
25
1.3 RESULTS AND
DISCUSSION.......................................................................................................
27
1.3.1 ACTIVATION CAPACITY OFLLERS, LEURS, VAIRS AND METRS TOWARDS
ALIPHATIC
FLUORINATED AMINO
ACIDS..........................................................................................................
27
1.3.2 GENERATION O F AN IN VIVO SYSTEM TO EVALUATE THE TRANSLATION O F
NCAAS IN RESPONSE TO
LIE
CODONS.................................................................................................................................
33
1.3.3 TESTING THE STRINGENCY O F THE ILERS TRANSLATIONAL SYSTEM
..........................................
38
1.3.4 EXPERIMENTS INDICATE THAT LEU IS, AMONG THE CAAS, ONE OF THE MAIN
THREATS FOR
ILERS FIDELITY IN RIBOSOMAL
TRANSLATION..................................................................................40
1.3.5 ILERS AND VAIRS DISPLAY PROOFREADING ACTIVITY AGAINST TFEGLY:
TFEGLY IS NOT
TRANSLATED WITH THE HELP O F ANY NATURAL
AARSS....................................................................
44
1.3.6 IN VIVO TRANSLATION O F TFEGLY USING THE POST-TRANSFER DEFECTIVE
MUTANT ILERS ALA 10
53
1.3.7 INVESTIGATING THE EFFECT OFLLERS POST-TRANSFER EDITING ON
TRANSLATION O F FLUORINATED
AMINO ACIDS (TFEGLY, TFLSFVAL, TFVAL, T FLLE)
.......................................................................
55
1.3.8 TOWARDS AN ILERS FOR SPECIFIC TRANSLATION O F MFEGLY, DFEGLY AND T
FEG LY
...........
58
1.3.9 *IDENTITY SWITCH* OF
TFLLE..............................................................................................60
1.4 CONCLUSION AND
OUTLOOK.....................................................................................................
63
2 PART I I
............................................................................................................................................65
2.1
INTRODUCTION......................................................................................................................
67
2.1.1 FUNCTIONS O F CYTOCHROME C
...........................................................................................
67
2.1.2 THE ROLE O FCY T C IN THE CELL RESPIRATION PRO CESS
........................................................
68
2.1.3 THE ROLE O FCY T C IN CELL
APOPTOSIS...............................................................................68
2.1.4 LOCAL ELECTRIC FIELDS AND VIBRATIONAL STARK EFFECT (V
SE)...........................................70
2.1.5 CYANO GROUP AS VSE REPORTER
.....................................................................................
71
2.1.6 FUNCTIONALIZATION O F CYTOCHROME C WITH NCA A S
.......................................................
71
2.1.7 HETERO TOGOUS EXPRESSION O F EUKARYOTIC CYT C IN E. C O
LI............................................72
2.2 AIM O F THIS
STUDY.................................................................................................................
75
2.3 RESULTS AND
DISCUSSION.......................................................................................................
77
2.3.1 INVESTIGATION O F FOCAL ELECTRIC FIELDS IN CYT C
.............................................................. 77
2.3.2 SENSING CONFORMATIONAL CHANGES IN CYT C
.....................................................................89
2.3.3 GLOBAL FLUORINATION O F CYT C USING THE SPI METHOD: TOWARDS
FINE-TUNING THE REDOX
POTENTIAL AND TOLERANCE TOWARD ORGANIC SOLVENTS
.................................................................
99
2.4 CONCLUSION AND
OUTLOOK...................................................................................................
105
3. MATE
HALS...................................................................................................................................
107
3.1 BUFFERS AND
SOLUTIONS.......................................................................................................
107
3.2
CHEMICALS............................................................................................................................110
3.3
ENZYMES..............................................................................................................................
110
3.4
KITS........................................................................................................................................I
L L
3.5
MEDIA...................................................................................................................................
I L L
3.6 PLASM
IDS..............................................................................................................................
112
3.7
STRAINS.................................................................................................................................
114
3.8 SYNTHETIC
OLIGONUCLEOTIDES...............................................................................................
115
3.9 TECHNICAL
EQUIPMENT.........................................................................................................115
4.
METHODS......................................................................................................................................119
4.1 MICROBIOLOGICAL
METHODS.................................................................................................
119
4.1.1 STORAGE AND CULTIVATION OFIS. COLI CELLS
.....................................................................
119
4.1.2 PREPARATION O F COMPETENT
CELLS...................................................................................
119
4.1.3
TRANSFORMATION.............................................................................................................
120
4.1.4 STANDARD GENE EXPRESSION IN E. C O
LI...........................................................................120
4.1.5 CELL
LYSIS........................................................................................................................121
4.1.6 SELECTIVE-PRESSURE INCORPORATION (SPI) O F NCAAS
....................................................
121
4.1.7 NCAA INCORPORATION VIA STOP CODON SUPPRESSION (S C S
)..........................................122
4.2 MOLECULAR BIOLOGICAL
METHODS.........................................................................................122
4.2.1 PLASMID DNA
ISOLATION...............................................................................................
122
4.2.2 RESTRICTION DIGESTION AND
LIGATION..............................................................................
123
4.2.3 SUBSTITUTION OFLLER S RESIDUES 241-250 BY 10 ALANINES
............................................
123
4.2.4 POLYMERASE CHAIN REACTION (PC R
)..........................................................................
124
4.3 BIOCHEMICAL
METHODS.......................................................................................................126
4.3.1 IMMOBILIZED METAL ANION CHROMATOGRAPHY (IM
AC)................................................126
4.3.2 ION EXCHANGE CHROMATOGRAPHY
(IEX)........................................................................
126
4.3.3 SIZE EXCLUSION CHROMATOGRAPHY (SE C
).....................................................................
127
4.3.4 REFOLDING OF PROTEINS FROM INCLUSION BO D IES
...........................................................
127
4.3.5 DIGESTFON WITH SUMO
PROTEASE.................................................................................127
4.3.6 ATP-PPJ EXCHANGE
ASSAY............................................................................................
128
4.3.7 AMINO ACYLATION
ASSAY.................................................................................................128
4.3.8 DEACYLATION
ASSAY........................................................................................................
129
4.3.9 AA-AMP SYNTHESIS ASSAY
..........................................................................................
129
4.4 ELECTROCHEMICAL
METHODS.................................................................................................129
4.4.1 SURFACE ENHANCED INFRARED ABSORPTION (SEIRA) SPECTROSCOPY
................................
129
4.4.2 CYCLIC VOLTAMMETRY (C V
)..........................................................................................
130
4.5
ANALYTICS.............................................................................................................................
130
4.5.1 AGAROSE GEL
ELECTROPHORESIS.......................................................................................
130
4.5.2 POLYACRYLAMIDE GEL ELECTROPHORESIS
..........................................................................
131
4.5.3 WESTERN B LO
T................................................................................................................131
4.5.4 MEASUREMENT O F PROTEIN AND DN A CONCENTRATION
....................................................
132
4.5.5 LIQUID CHROMATOGRAPHY-ELECTROSPRAY IONIZATION-MASS SPECTROMETRY
(LC-ESI-MS)
132
4.5.6 ULTRAVIOLET-VISIBLE (UV-VIS)
SPECTROSCOPY..............................................................133
4.5.7 CIRCULAR DIEHROISM (CD)
SPECTROSCOPY.....................................................................
133
4.5.8 INFRARED ABSORPTION (ER)
SPECTROSCOPY......................................................................
133
4.5.9 RESONANCE RAMAN (RR)
SPECTROSCOPY......................................................................
134
4.5.9
,9F-NMR......................................................................................................................
134
LIST O F
ABBREVIATIONS.................................................................................................................135
5.
REFERENCES.................................................................................................................................141
6.
APPENDIX...................................................................................................................................159
6.1 NUCLEOTIDE
SEQUENCES.....................................................................................................
159
6.2 AMINO ACID
SEQUENCES....................................................................................................160
6.3 SDS-PAGE GEL O F PURIFIED A A R
SS..............................................................................163
6.4 IN VIVO TRANSLATION USING ILERS MUTANTS T243R AND T242P AND IN VITRO
POST-TRANSFER
EDITING AGAINST
TFEGLY..........................................................................................................
164
7. EIDESSTATTLICHE ERKLAERUNG
167
|
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| language | English |
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| spelling | Völler, Jan-Stefan Verfasser aut Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields by Jan-Stefan Völler Berlin 2016 IV, 167 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Freie Universität Berlin 2016 Aminosäuren (DE-588)4142205-3 gnd rswk-swf Proteinsynthese (DE-588)4261878-2 gnd rswk-swf Peptidsynthese (DE-588)4173715-5 gnd rswk-swf Fluorierung (DE-588)4268617-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Aminosäuren (DE-588)4142205-3 s Fluorierung (DE-588)4268617-9 s Proteinsynthese (DE-588)4261878-2 s Peptidsynthese (DE-588)4173715-5 s DE-188 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029177270&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Völler, Jan-Stefan Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields Aminosäuren (DE-588)4142205-3 gnd Proteinsynthese (DE-588)4261878-2 gnd Peptidsynthese (DE-588)4173715-5 gnd Fluorierung (DE-588)4268617-9 gnd |
| subject_GND | (DE-588)4142205-3 (DE-588)4261878-2 (DE-588)4173715-5 (DE-588)4268617-9 (DE-588)4113937-9 |
| title | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields |
| title_auth | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields |
| title_exact_search | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields |
| title_full | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields by Jan-Stefan Völler |
| title_fullStr | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields by Jan-Stefan Völler |
| title_full_unstemmed | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields by Jan-Stefan Völler |
| title_short | Activation, proofreading and translation of aliphatic fluorinated amino acids by class Ia AARSs |
| title_sort | activation proofreading and translation of aliphatic fluorinated amino acids by class ia aarss modification of cytochrome c with non canonical amino acids to enable spectroscopic investigation of protein states and local electric fields |
| title_sub | modification of cytochrome c with non-canonical amino acids to enable spectroscopic investigation of protein states and local electric fields |
| topic | Aminosäuren (DE-588)4142205-3 gnd Proteinsynthese (DE-588)4261878-2 gnd Peptidsynthese (DE-588)4173715-5 gnd Fluorierung (DE-588)4268617-9 gnd |
| topic_facet | Aminosäuren Proteinsynthese Peptidsynthese Fluorierung Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029177270&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT vollerjanstefan activationproofreadingandtranslationofaliphaticfluorinatedaminoacidsbyclassiaaarssmodificationofcytochromecwithnoncanonicalaminoacidstoenablespectroscopicinvestigationofproteinstatesandlocalelectricfields |