Biochemical characterization of Neurospora crassa nitrate reductase:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 82 S. Ill., graph. Darst. |
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| adam_text | BIOCHEMICAL CHARACTERIZATION OF NEUROSPORA CRASSA NITRATE REDUCTASE
VON DER FAKULTAT FUR LEBENSWISSENSCHAFTEN
DER TECHNISCHEN UNIVERSITAT CAROLO-WILHELMINA ZU BRAUNSCHWEIG
ZUR ERLANGUNG DES GRADES EINES
DOKTORS DER NATURWISSENSCHAFIEN
(DR. RER. NAT.)
GENEHMIGTE
DISSERTATION
VON
AUS
PHILLIP RINGEL
WOLFE
RIBUTTEL
HTTP://D-NB.INFO/1054935610
PHILLIP RINAEL
TABLE OF CONTENTS
TABLE OF CONTENTS
1 INTRODUCTION
1.1 BIOLOGICAL IMPORTANCE
OF MOLYBDENUM: THE MOLYBDENUM COFACTOR
1.2 BIOSYNTHESIS OF THE MOLYBDENUM
COFACTOR IN EUKARYOTES
1.3 MOLYBDENUM COFACTOR DEPENDENT ENZYMES
1.4 EUKARYOTIC NITRATE REDUCTASE
1.5 NEUROSPORA CRASSA NITRATE NONUTILIZER MUTANTS
LINK NITRATE ASSIMILATION AND
MOLYBDENUM METABOLISM
1.6 AIMS OF THIS
STUDY
2 RESULTS
2.1 BIOCHEMICAL AND
MOLECULAR STUDIES OF MOLYBDENUM COFACTOR INSERTION
INTO
NEUROSPORA CRASSA NITRATE
REDUCTASE
2.1.1 CONSTRUCTION AND
HETEROLOGOUS EXPRESSION OF RECOMBINANT N. CRASSA NR
2.1.2 PURIFICATION OF RECOMBINANT
NEUROSPORA CRASSA NR
2.1.3 PROSTHETIC GROUP
CONTENT OF RECOMBINANT
NR
2.1.4 NITRATE AND
CYTOCHROME C REDUCING
ACTIVITIES OF RECOMBINANT NR VARIANTS
2.1.5 OLIGOMERIZATION STATE OF RECOMBINANT NEUROSPORA NR
2.1.6 IN VITRO INCORPORATION
OF MOCO INTO
N. CRASSA APO-NR
2.1.7 ELECTRON PARAMAGNETIC RESONANCE
STUDIES OF RECONSTITUTED NEUROSPORA
NR
2.1.8 MOCO-DEPENDENT DIMERIZATION OF MOCO
FREE NR
2.1.9 QUANTIFICATION OF MOCO
TRANSFER
2.1.10 THE SEQUENCE OF PROSTHETIC
GROUP INSERTION INTO NEUROSPORA NR
2.2 GENETIC CHARACTERIZATION
OF THE NEUROSPORA CRASSA MOLYBDENUM
COFACTOR
BIOSYNTHESIS
2.2.1 PRESENT ANNOTATION OF NEUROSPORA
CRASSA MOCO
BIOSYNTHESIS GENES
2.2.2 FUNCTIONAL COMPLEMENTATION
OF N. CRASSA MOCO
BIOSYNTHESIS MUTANTS
2.2.3 SEQUENCING OF THE N. CRASSA
MOCO BIOSYNTHESIS MUTANTS
1
1
3
5
6
7
8
9
9
9
9
11
12
14
17
19
20
21
23
27
27
28
29
PHILLIP RINAEL
TABLE OF CONTENTS
2.2.4
CREATION AND CHARACTERIZATION OF MOCO BIOSYNTHESIS KNOCK OUT STRAINS
3 DISCUSSION
3.1 IN VITRO STUDIES OF MOCO
INSERTION INTO N. CRASSA NR
3.1.1 HETEROLOGOUS EXPRESSION, PURIFICATION AND PROSTHETIC GROUPS OF N.
CRASSA NR
3.1.2 ENZYMATIC ACTIVITIES OF N. CRASSA NR VARIANTS
3.1.3 RECONSTITUTION STUDIES ON N. CRASSA MOCO-FREE NR
3.1.4 N. CRASSA NR OLIGOMERIZATION IS DEPENDENT ON THE PYRANOPTERIN
BACKBONE
3.1.5 STRUCTURAL BASIS OF MOCO-INDUCED NR DINNER FORMATION
3.2 CHARACTERIZATION OF THE N. CRASSA MOCO BIOSYNTHESIS
3.2.1 N. CRASSA N/F-MUTANTS:
UNDERLYING MUTATIONS AND FUNCTIONAL COMPLEMENTATION
3.2.2 CREATION AND CHARACTERIZATION OF N. CRASSA MOCO BIOSYNTHESIS KNOCK
OUT
STRAINS
3.3 OUTLOOK
4 MATERIAL AND METHODS
4.1 MATERIAL
4.1.1 CHEMICALS AND ENZYMES
4.1.2 MEDIA, BUFFERS AND SOLUTIONS
4.1.3 ORGANISM STRAINS
4.1.4 VECTORS
4.1.5 MEDIA AND STOCK SOLUTIONS
4.1.6 ANTIBODIES
4.1.7 OLIGONUCLEOTIDES
4.1.8 EQUIPMENT
4.2 METHODS
4.2.1 MOLECULAR BIOLOGICAL METHODS
4.2.1.1 PREPARATION OF PLASMID
DNA
4.2.1.2 RESTRICTION ENDONUCLEASE DIGESTION OF DNA
32
35
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36
36
37
38
39
40
41
41
43
43
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45
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50
51
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51
PHILLIP RINOEL
TABLE OF CONTENTS
4.2.1.3 LIGATION
51
4.2.1.4 GEL ELECTROPHORESIS OF DNA
51
4.2.1.5 DNA ELUTION FROM
AGAROSE GELS 52
4.2.1.6 POLYMERASE CHAIN
REACTION (PCR) 52
4.2.1.7 OVERLAP EXTENSION PCR (OE-PCR)
53
4.2.1.8 SITE DIRECTED MUTAGENESIS
53
4.2.1.9 REVERSE TRANSCRIPTASE-PCR(RT-PCR)
53
4.2.1.10 CULTIVATION OF E.
COLI
53
4.2.1.11 STORAGE OF E. COLI
54
4.2.1.12 PREPARATION OF COMPETENT
E. COLI
54
4.2.1.13 TRANSFORMATION INTO E. COLI
54
4.2.1.14 AUTOMATED DNA SEQUENCE
ANALYSIS
55
4.2.2 WORKING WITH NEUROSPORA CRASSA
55
4.2.2.1 N. CRASSA MEDIA,
GROWTH CONDITIONS AND STORAGE
55
4.2.2.2 IDENTIFICATION OF N. CRASSA
MOCO BIOSYNTHESIS GENES
55
4.2.2.3 ISOLATION OF GENOMIC
DNA FROM N. CRASSA 56
4.2.2.4 TRANSFORMATION INTO N. CRASSA VIA ELECTROPORATION 56
4.2.2.5 CREATION OF N.
CRASSA KNOCK OUT
STRAINS 57
4.2.2.6 CROSSING
57
4.2.2.7 PROTEIN AND
METABOLITE EXTRACTION
FROM N. CRASSA 58
4.2.3 BIOCHEMICAL METHODS 58
4.2.3.1 EXPRESSION OF RECOMBINANT
PROTEINS IN E. COLI 58
4.2.3.2 E. COLI CELL
LYSIS AND PROTEIN
EXTRACTION
59
4.2.3.3 AFFINITY PURIFICATION
OF 6XHIS- AND
TWINSTREP-TAGGED PROTEINS 59
4.2.3.4 DETERMINATION OF PROTEIN
CONCENTRATIONS
59
4.2.3.5 BUFFER EXCHANGE OF PROTEIN SOLUTIONS 60
4.2.3.6 SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL
ELECTROPHORESIS (SDS-PAGE) 60
4.2.3.7 SEMI-DRY IMMUNE BLOT 60
4.2.3.8 DETECTION OF TWINSTREP-TAGGED PROTEINS 61
PHILLIP RINAEL
TABLE OF CONTENTS
4.2.3.9 AKTA GEL FILTRATION 61
4.2.3.10 NADPH-DEPENDENT NITRATE REDUCTASE ACTIVITY 62
4.2.3.11 NADPH-DEPENDENT CYTOCHROME
C REDUCTASE ACTIVITY 62
4.2.3.12 IN VITRO
RECONSTITUTION OF MOCO-FREE NITRATE REDUCTASE 62
4.2.3.13 CIRCULAR DICHROISM
(CD) SPECTROSCOPY 63
4.2.3.14 ANALYTICAL ULTRACENTRIFUGATION 63
4.2.3.15 ELECTRON PARAMAGNETIC RESONANCE (EPR) SPECTROSCOPY 63
4.2.3.16 MASS SPECTROMETRY (MS) 63
4.2.3.17 QUANTIFICATION OF CPMP, MOCO OR MPT VIA HPLC 64
4.2.3.18 QUANTIFICATION OF HEME 64
4.2.3.19 INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS) 64
4.2.3.20 ISOTHERMAL TITRATION CALORIMETRY (ITC) 65
5 LITERATURE 66
6
SUMMARY
82
|
| any_adam_object | 1 |
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| author_facet | Ringel, Phillip |
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| author_sort | Ringel, Phillip |
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| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.79129567 |
| dewey-search | 572.79129567 |
| dewey-sort | 3572.79129567 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Ringel, Phillip Verfasser (DE-588)1055021787 aut Biochemical characterization of Neurospora crassa nitrate reductase von Phillip Ringel 2014 82 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Braunschweig, Techn. Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027574536&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ringel, Phillip Biochemical characterization of Neurospora crassa nitrate reductase |
| subject_GND | (DE-588)4113937-9 |
| title | Biochemical characterization of Neurospora crassa nitrate reductase |
| title_auth | Biochemical characterization of Neurospora crassa nitrate reductase |
| title_exact_search | Biochemical characterization of Neurospora crassa nitrate reductase |
| title_full | Biochemical characterization of Neurospora crassa nitrate reductase von Phillip Ringel |
| title_fullStr | Biochemical characterization of Neurospora crassa nitrate reductase von Phillip Ringel |
| title_full_unstemmed | Biochemical characterization of Neurospora crassa nitrate reductase von Phillip Ringel |
| title_short | Biochemical characterization of Neurospora crassa nitrate reductase |
| title_sort | biochemical characterization of neurospora crassa nitrate reductase |
| topic_facet | Hochschulschrift |
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