Biochemical characterisation of photosystem I-complexes in diatoms [Elektronische Ressource] / von Thomas Veith

Thomas Veith Pdfcreator

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 12
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Biochemical Characterisation of
Photosystem I Complexes in Diatoms
Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften
vorgelegt beim Fachbereich Biowissenschaften der
Johann Wolfgang Goethe – Universität
in Frankfurt am Main
von
Thomas Veith
aus Frankfurt am Main
Frankfurt 2009
(D30)vom Fachbereich .............................................................. der
Johann Wolfgang Goethe – Universität als Dissertation angenommen.
Dekan: ...........................................................................
Gutachter:........................................................................
Datum der Disputation:............................................................SturzbomberDerkeinGroschenistCONTENTS v
Contents
Abbreviations viii
1 Introduction 1
1.1 Oxygenic photosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Structure of Photosystem I . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3 Regulation of excitation energy distribution and electron flow . . . . . . 11
1.4 Aspects of photosynthesis in diatoms . . . . . . . . . . . . . . . . . . . 14
1.5 Aims of this work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
2 Materials and methods 20
2.1 Biological material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.2.1 Phaeodactylum tricornutum . . . . . . . . . . . . . . . . . . . . 20
2.2.2 Cyclotella meneghiniana . . . . . . . . . . . . . . . . . . . . . . 21
2.2.3 Pisum sativum . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2.4 Synechocystis sp. PCC 6803 . . . . . . . . . . . . . . . . . . . 21
2.3 Isolation of thylakoids . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.3.1 P. tricornutum . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.2 C. meneghiniana . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.3.3 P. sativum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3.4 Synechocystis sp. PCC 6803 . . . . . . . . . . . . . . . . . . . 23
2.4 Purification of pigment-protein complexes . . . . . . . . . . . . . . . . . 24
2.4.1 Ion exchange chromatography (IEX) . . . . . . . . . . . . . . . 24
2.4.2 Discontinuous sucrose gradient centrifugation . . . . . . . . . . . 25
2.4.3 Analytical gel filtration . . . . . . . . . . . . . . . . . . . . . . . 25
2.4.4 Concentration & washing of isolated pigment-protein fractions . . 26vi CONTENTS
2.5 Gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.5.1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.5.2 Blue native-PAGE (BN-PAGE) . . . . . . . . . . . . . . . . . . 27
2.5.3 Coomassie staining . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.5.4 Silver staining . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.6 Protein transfer & immunodetection . . . . . . . . . . . . . . . . . . . 29
2.6.1 Western Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.6.2 Slot Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.6.3 Immunodetection . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.7 Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.7.1 Absorbance spectra . . . . . . . . . . . . . . . . . . . . . . . . 31
2.7.2 Determination of P700 . . . . . . . . . . . . . . . . . . . . . . . 31
2.7.3 Chlorophyll determination . . . . . . . . . . . . . . . . . . . . . 31
2.7.3.1 Chlorophyll c containing algae . . . . . . . . . . . . . 31
2.7.3.2 Higher plants . . . . . . . . . . . . . . . . . . . . . . 32
2.7.3.3 Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . 32
2.7.4 Fluorescence spectra . . . . . . . . . . . . . . . . . . . . . . . . 32
2.8 High Performance Liquid Chromatography (HPLC) . . . . . . . . . . . . 33
2.9 Electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.10 Sequence comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3 Results 35
3.1 A monomeric PS I-FCP complex of P. tricornutum . . . . . . . . . . . . 35
3.1.1 Isolation of a PS I complex from P. tricornutum . . . . . . . . . 35
3.1.2 Polypeptide composition of the PS I complex . . . . . . . . . . . 36
3.1.3 Characterisation of the complex . . . . . . . . . . . . . . . . . . 38
3.1.4 Higher organisation of the PS I-FCP complex . . . . . . . . . . . 45CONTENTS vii
3.1.5 Preparation yield . . . . . . . . . . . . . . . . . . . . . . . . . . 50
3.1.6 Analysis of the IEX fractions II and V . . . . . . . . . . . . . . . 50
3.1.7 Purification of a PS I complex by discontinuous sucrose gradient
centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3.2 Association of Fcp polypeptides with PS I in the diatom C. meneghiniana 55
3.2.1 Isolation of pigment-protein complexes . . . . . . . . . . . . . . 55
3.2.2 Polypeptide analysis . . . . . . . . . . . . . . . . . . . . . . . . 60
3.2.3 Further purification of PS I . . . . . . . . . . . . . . . . . . . . 62
4 Discussion 67
4.1 The monomeric PS I-FCP complex of P. tricornutum . . . . . . . . . . . 68
4.2 Association of Fcp polypeptides with PS I of C. meneghiniana . . . . . . 76
4.3 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
5 Summary 86
6 Zusammenfassung 89
7 Acknowledgements 112
8 Curriculum vitae 114viii CONTENTS
Abbreviations
Å .................... Ångstrom
APS ................. ammonium persulfate
ATP ................. adenosine triphosphate
B1 ................... buffer 1
BChl ................. bacteriochlorophyll
Bis-Tris .............. 2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol
BN-PAGE ............ Blue native-polyacrylamide gel electrophoresis
C .................... concentration [%] of bisacrylamide in relation to T, see T
cab proteins .......... Chl ab-binding proteins, see Chl
CET ................. cyclic electron transport
Chl .................. chlorophyll
cyt c ................ cytochrome c6 6
Da ................... Dalton
DDM ................ n-dodecyl b-D-maltoside
ECL ................. enhanced chemiluminescence
EDTA ................ ethylendiamintetraacetic acid
fcp .................. genes encoding fucoxanthin chlorophyll proteins
FCP ................. a complex of fucoxanthin chlorophyll proteins
Fcp .................. fucoxanthin chlorophyll proteins
+ +FNR ................. ferredoxin:NADP oxidoreductase, see NADP
HB .................. homogenisation buffer
HEPES .............. 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
HPLC ................ high performance liquid chromatography
IEX .................. ion exchange chromatography
kDa .................. kilo Dalton, 1000 DaltonCONTENTS ix
l .................... wavelength in nm
LET ................. linear electron transport
LHC ................. light-harvesting complexes
LWC ................. longwavelength chlorophyll
MALDI .............. matrix supported laser desorption/ ionisation
Mes .................. 4-Morpholineethanesulfonic acid
MS .................. mass spectrometry
MW ................. molecular weight
NAD ................. nicotinamide adenine dinucleotide
NG .................. nonyl b-D-glucopyranoside
NADP ............... nicotinamide adenine dinucleotide phosphate
NPQ ................. non-photochemical quenching
OG .................. octyl b-D-glucopyranoside
p.a. .................. per analysi
PC .................. plastocyanin
PQ .................. plastoquinone
PS I ................. photosystem I
PS II ................. II
Psa .................. PS I subunit
Psb .................. PS II subunit
psi ................... pounds per square inch
PVDF ................ Polyvinylidene difluoride
Q .................... quinone
qE ................... energy dependent fluorescence quenching
qI .................... photoinhibition fluorescence quenching
qT ................... state transition fluorescence
RB .................. resuspension bufferx CONTENTS
RC .................. reaction centre
rpm .................. rounds per minute
rRNA ................ ribosomal ribonucleic acid
RT .................. room temperature
S .................... Svedberg
SB ................... sample buffer
SDS ................. Sodium dodecyl sulfate
SDS-PAGE ........... Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SU ................... subunit
T .................... concentration [%] of acrylamide and bisacrylamide
TEMED .............. N, N, N’, N’-tetra-methylethylendiamine
TOF ................. time of flight
Tris .................. Tris(hydroxymethyl)aminomethane
TX 100 .............. Triton X 100
WB .................. washing bufferLIST OF FIGURES xi
List of Figures
1 The light reaction of oxygenic photosynthesis . . . . . . . . . . . . . . . 4
2 Photosystem I complexes of cyanobacteria and higher plants . . . . . . . 9
3 General structure of a pennate diatom . . . . . . . . . . . . . . . . . . 14
4 IEX of solubilised thylakoids of P. tricornutum . . . . . . . . . . . . . . 36
5 Western Blot of IEX fractions III – V . . . . . . . . . . . . . . . . . . . 37
6 SDS-P