Biochemical and biotechnological approaches as basis for structure determination of pigment-protein complexes of oxygenic photosynthesis [Elektronische Ressource] / von Holger Fey

Tyler Durdon

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2006
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Biochemical And Biotechnological Approaches As Basis
For Structure Determination Of Pigment-Protein
Complexes Of Oxygenic Photosynthesis



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich Biowissenschaften
der Johann Wolfgang Goethe – Universität
in Frankfurt am Main





von
Holger Fey
aus Pirmasens



Frankfurt am Main 2006
(D 30)












vom Fachbereich ……………………………………………………………………...der

Johann Wolfgang Goethe – Universität als Dissertation angenommen.












Dekan: ……………………………………………………………………………………

Gutachter: …...……………………………………………………………………………

Datum der Disputation: ......………………………………………………………………














“I may not have gone where I intended to go,
but I think I have ended up where I needed to be.”

Douglas Adams
















Contents

Abbreviations .................................................................................................................... iv
Figure index ....................................................................................................................... v
Table index........................................................................................................................ vi

I. Introduction ........................................................................................ 1

1. Photosynthesis ........................................................................................... 1

2. Light-harvesting and energy transfer.......................................................... 7

3. Structure and function of photosystem II ................................................. 11

4. Aims of this work ...................................................................................... 15


II. Materials and Methods ..................................................................... 17

1. Materials................................................................................................... 17
1.1 Biological material ..................................................................................................... 17
1.2 Plasmid DNA and primers ......................................................................................... 18
1.3 Restriction enzymes 19

2. Methods .................................................................................................... 20
2.1 Plasmid DNA preparation .......................................................................................... 20
2.2 Mutagenesis through altered primers in PCR ............................................................ 21
2.3 Restriction of DNA 22
2.4 Agarose gel electrophoresis and gel extraction of DNA bands ................................. 23
2.5 Polyacrylamide gel electrophoresis for small DNA fragments.................................. 24
2.6 Ligation of DNA ........................................................................................................ 25
2.7 Transformation of Escherichia coli ........................................................................... 25
2.8 Transformation and shoot regeneration of Nicotiana tabacum 27
2.9 Growth and culture of tobacco plants ........................................................................ 31
2.10 Thylakoid preparation .............................................................................................. 31
2.11 Photosystem II preparation by solubilisation and centrifugation............................. 33
2.12 Photosystem II preparation by affinity chromatography ......................................... 33
2.13 Chlorophyll determination (Chl a + Chl b).............................................................. 35
2.14 Absorption spectroscopy.......................................................................................... 35
2.15 Polyacrylamide gel electrophoresis of proteins ....................................................... 35
2.16 Western blot ............................................................................................................. 37
2.17 Oxygen evolution..................................................................................................... 38
2.18 Pulse amplitude modulated fluorescence measurement (PAM) .............................. 38
2.19 Two-dimensional crystallisation of photosystem II................................................. 39
2.20 Electron microscopy and sample preparation .......................................................... 39
2.21 FCP preparation from Cyclotella meneghiniana...................................................... 40
2.22 Chlorophyll determination in 90% acetone (Chl a + Chl c) .................................... 41
2.23 Pigment determination by High Performance Liquid Chromatography .................. 42
i







III. Results ........................................................................................... 43

1. Transformation of Nicotiana tabacum ....................................................... 43
-1.1 Vector preparation (pbKS+SacI )............................................................................... 44
-1.2 Cloning psbE (pbKS+SacI psbE) 46
-1.3 Inserting His-tags (pbKS+SacI psbE-His )............................................................. 47 6/10
-1.4 Inserting the resistance cassette (pbKS+SacI psbE-His -aadA) ............................. 49 6/10
1.5 Biolistic transformation of tobacco chloroplasts........................................................ 50

2. Characterisation of transgenic tobacco ..................................................... 52
2.1 Chlorophyll content of tobacco leafs ......................................................................... 52
2.2 Oxygen evolution of tobacco thylakoids.................................................................... 53
2.3 Pulse-amplitude modulated (PAM) fluorometry ....................................................... 54

3. Preparation of photosystem II.................................................................. 55
3.1 Preparation of photosystem II from different tobacco strains.................................... 55
3.2 His -tag facilitated photosystem II preparation.......................................................... 56 6
3.3 His -tag facilitated phot ........................................................ 58 10
3.4 Wildtype control photosystem ............................................................ 59
3.5 Protein composition of different column fractions .................................................... 60
3.6 Two-dimensional crystallisation of photosystem II................................................... 63

4. Characterisation of fucoxanthin-chlorophyll-proteins ............................... 64


IV. Discussion ....................................................................................... 67

1. Photosystem II ......................................................................................... 67

2. Energy transfer in fucoxanthin-chlorophyll-proteins................................. 74

3. Outlook ..................................................................................................... 79


V. Summary .......................................................................................... 81


VI. Zusammenfassung .......................................................................... 85


VII. References..................................................................................... 91

ii





VIII. Appendix .................................................................................... 101

1. Equipment and chemicals ....................................................................... 101
1.1 Equipment ................................................................................................................ 101
1.2 Chemicals................................................................................................................. 102

2. Sequences............................................................................................... 106
-2.1 pbKS+SacI psbE-His NC (EH1).............................................................................. 107 6
-2.2 pbKS+SacI NC (EH2) ............................................................................ 108 10
-2.3 pbKS+SacI psbE-His C (EH3)................................................................................. 109 6
-2.4 pbKS+SacI C (EH4) ............................................................................... 11010

3. HPLC parameters .................................................................................... 111
3.1 HPLC retention times 111
3.2 HPLC calibration factors and calibration