Crystallisation and functional studies with ethylene receptor ETR1 [Elektronische Ressource] / Elisa Buchen. Gutachter: Dieter Willbold. Betreuer: Georg Groth

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Crystallisation and functional studies with ethylene receptor ETR1 Inaugural-Dissertation Elisa Buchen Crystallisation and functional studies with ethylene receptor ETR1 Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Elisa Buchen aus Remscheid Düsseldorf, Februar 2011 Aus dem Institut für Biochemie der Pflanzen der Heinrich-Heine-Universität Düsseldorf Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. G. Groth Koreferent: Prof. Dr. D. Willbold Tag der mündlichen Prüfung: 14.04.2011 Titelbild: Arabidopsis thaliana Ökotyp Landsberg erecta. Die Pflanze wurde freundlicherweise zur Verfügung gestellt von Helge Pallakies, Institut für Genetik der Universität Düsseldorf. Contents1 Abstract..........................................................................................................7 2 Zusammenfassung...........................................................................................9 3 Introduction.................................................................................................. 11 3.1 Plant hormones: Communication is all!............................................................................
Publié le : samedi 1 janvier 2011
Lecture(s) : 68
Source : D-NB.INFO/1015435173/34
Nombre de pages : 176
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Crystallisation and functional studies
with ethylene receptor ETR1
Inaugural-Dissertation
Elisa Buchen






Crystallisation and functional studies
with ethylene receptor ETR1







Inaugural-Dissertation


zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf









vorgelegt von


Elisa Buchen
aus Remscheid




Düsseldorf, Februar 2011


Aus dem Institut für Biochemie der Pflanzen
der Heinrich-Heine-Universität Düsseldorf



















Gedruckt mit der Genehmigung der
Mathematisch-Naturwissenschaftlichen Fakultät der
Heinrich-Heine-Universität Düsseldorf



Referent: Prof. Dr. G. Groth
Koreferent: Prof. Dr. D. Willbold

Tag der mündlichen Prüfung: 14.04.2011


Titelbild: Arabidopsis thaliana Ökotyp Landsberg erecta. Die Pflanze wurde freundlicherweise
zur Verfügung gestellt von Helge Pallakies, Institut für Genetik der Universität Düsseldorf.

Contents
1 Abstract..........................................................................................................7
2 Zusammenfassung...........................................................................................9
3 Introduction.................................................................................................. 11
3.1 Plant hormones: Communication is all!.................................................................................11
3.2 The plant hormone ethylene.................................................................................................11
3.2.1 The role of ethylene in plant life cycle....................................................................12
3.2.2 Ethylene biosynthesis.............................................................................................12
3.2.3 The ethylene signalling pathway in Arabidopsis thaliana ........................................13
3.2.4 Cross-talk between ethylene and other plant hormones...........................................19
3.2.5 Objectives of this thesis..........................................................................................20
4 Materials & Methods.................................................................................... 21
4.1 Materials..............................................................................................................................21
4.1.1 Equipment .............................................................................................................21
4.1.2 Chromatography supplies.......................................................................................22
4.1.3 Crystallographic supplies........................................................................................22
4.1.4 Kits........................................................................................................................23
4.1.5 Filters and Membranes...........................................................................................23
4.1.6 Chemicals and Buffers............................................................................................23
4.1.7 Radiochemicals.......................................................................................................24
4.1.8 Enzymes ................................................................................................................24
4.1.9 Antibodies..............................................................................................................25
4.1.10 Oligonucleotides.....................................................................................................25
4.1.11 Custom gene synthesis ...........................................................................................27
4.1.12 Vectors ..................................................................................................................27
4.1.13 Bacterial strains: Escherichia coli...........................................................................29
4.2 Microbiological methods .......................................................................................................30
4.2.1 Media for cultivation of E. coli cells.......................................................................30
4.2.2 Preparation and transformation of chemically competent E. coli cells.....................30
4.3 Molecular biological methods................................................................................................31
4.3.1 Amplification and isolation of plasmid DNA from E. coli .......................................31
4.3.2 Determination of DNA concentration .....................................................................31
4.3.3 Polymerase chain reaction to amplify DNA ............................................................31
4.3.4 Agarose gel electrophoresis.....................................................................................32
4.3.5 Cloning, Mutagenesis and Sequencing.....................................................................33
4.3.6 SLIC: Sequence and ligation independent cloning ...................................................34
4.4 Protein biochemical methods................................................................................................35
4.4.1 Protein quantification.............................................................................................35
4.4.2 SDS polyacrylamide gel electrophoresis...................................................................36
4.4.3 Silver staining of SDS-PAGE gels...........................................................................36
4.4.4 Western blotting ....................................................................................................37
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4.4.5 Dot blotting...........................................................................................................37
4.4.6 Immunodetection ...................................................................................................38
4.4.7 Proteolytic cleavage ...............................................................................................38
4.5 Expression of recombinant proteins in E. coli .......................................................................39
4.5.1 Expression of full-length receptor protein AtETR1.................................................39
4.5.2 Expression of the extramembrane domain of AtETR1 ............................................40
4.5.3 Expression of PpETR1 and LeETR1......................................................................40
4.6 Purification of recombinant proteins from E. coli – general methods.....................................41
4.6.1 Cell disruption .......................................................................................................41
4.6.2 Isolation and solubilisation of E. coli membranes....................................................41
4.6.3 Isolation and purification of inclusion bodies ..........................................................42
4.6.4 Solubilisation of inclusion bodies ............................................................................42
4.6.5 Refolding of proteins solubilised from inclusion bodies............................................43
4.6.6 Size exclusion chromatography...............................................................................43
4.6.7 Removal of DnaK contamination............................................................................44
4.7 Purification protocols for individual proteins ........................................................................45
4.7.1 Purification of full-length receptor protein AtETR1................................................45
4.7.2 Purification of the extramembrane domain of AtETR1...........................................47
4.7.3 Purification of full-length PpETR1 and LeETR1....................................................48
4.7.4 Preparation of truncated PpETR1 and LeETR1 from inclusion bodies ...................48
4.8 Protein characterisation........................................................................................................49
4.8.1 In vitro kinase assay...............................................................................................49
4.8.2 Circular dichroism spectroscopy .............................................................................50
4.8.3 Fluorescence spectroscopy......................................................................................50
314.8.4 P-NMR spectroscopy............................................................................................51
4.8.5 Protein crystallography..........................................................................................52
4.8.6 Bioinformatics methods and software tools.............................................................57
5 Part A: Functional studies on ethylene receptor ETR1 from A. thaliana.. 59
5.1 Intrinsic tryptophan fluorescence to resolve ligand binding and conformational changes in
ETR1...................................................................................................................................60
5.1.1 Cloning of single tryptophan substitution mutants .................................................61
5.1.2 Expression and purification of recombinant ETR1..................................................64
5.1.3 Characterisation of single tryptophan substitution mutants....................................65
5.2 Ligand binding to ethylene receptor ETR1 ...........................................................................71
5.2.1 Binding of copper cofactor and ethylene.................................................................71
5.2.2 Characterisation of the quenching effect.................................................................74
5.2.3 Effect of His-tag on copper binding ........................................................................78
5.3 Characterisation of the ETR1 phosphorylation state.............................................................80
5.3.1 Conformational changes upon phosphorylation of ETR1 probed by engineered Trp
reporter groups ......................................................................................................81
315.3.2 P-NMR spectroscopy to assign the phosphorylation sites in ETR1 .......................86
5.4 Synopsis...............................................................................................................................89

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6 Part B: Crystallisation of ETR1 from A. thaliana ...................................... 91
6.1 Crystallisation of the extra membrane domain of ETR1........................................................92
6.1.1 Optimising crystallisation conditions ......................................................................94
6.1.2 Protein modification............................................................................................. 100
6.2 Crystallisation of full-length ETR1..................................................................................... 107
6.2.1 Effect of histidine affinity tag............................................................................... 108
6.2.2 Influence of the purification method on crystallisation of ETR1............................ 109
6.3 Synopsis............................................................................................................................. 115
7 Part C: Crystallisation of ETR1 orthologs from P. patens and
L. esculentum.............................................................................................. 117
7.1 Cloning and expression of receptor constructs..................................................................... 119
7.1.1 Cloning of receptor constructs.............................................................................. 120
7.1.2 Expression studies................................................................................................ 120
7.2 Purification and characterisation of PpETR1 and LeETR1................................................. 126
7.2.1 Receptor constructs lacking the membrane domain............................................... 126
7.2.2 Full-length receptor constructs............................................................................. 135
7.3 Crystallisation studies ........................................................................................................ 139
7.3.1 Full-length ethylene receptor ETR1 from L. esculentum....................................... 139
7.3.2 Full-length ethylene receptor ETR1 from P. patens.............................................. 140
7.4 Synopsis............................................................................................................................. 141
7.4.1 Perspective on further crystallisation studies ........................................................ 142
7.4.2 Taxonomic distribution of ethylene receptors in kingdom Viridiplantae ................ 143
7.4.3 Secondary structure analysis of orthologs AtETR1, PpETR1 and LeETR1........... 145
8 Concluding remarks.................................................................................... 149
9 Appendix..................................................................................................... 151
9.1 Abbreviations..................................................................................................................... 151
9.2 Maps of expression vectors ................................................................................................. 153
9.3 List of detergents used in this thesis ................................................................................... 158
9.4 Refolding buffers ................................................................................................................ 159
10 References................................................................................................... 161
11 Acknowledgements ..................................................................................... 171
12 Erklärung.................................................................................................... 173
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Abstract
1 Abstract
The gas ethylene has a wide influence on various physiological and developmental processes
within plants including senescence and abscission of petals, leafs and fruits. The plant hormone
is also known as a mediator of biotic and abiotic stress responses upon flooding, drought or
pathogen infection. Genetic studies in the model plant Arabidopsis thaliana revealed that
binding of ethylene occurs at specific receptor proteins located at the endoplasmic reticulum.
To ultimately understand how these membrane proteins perceive and transmit the ethylene
signal throughout a complex network and to elucidate potential interactions with other proteins
of the signalling cascade, it is essential to analyse and characterise the receptors at the protein
level.

Within the first part of this thesis, monitoring of intrinsic tryptophan fluorescence was used in
addition to a radioactive assay to characterise ligand binding and phosphorylation events in the
ethylene receptor protein ETR1 from Arabidopsis thaliana. Fluorescence based studies indicated
interaction of the receptor protein with the copper cofactor necessary for binding of ethylene.
Phosphorylation experiments revealed that phosphorylation can occur on further residues in the
kinase and receiver domain of the receptor other than the putative sites H353 and D659. In this
31context, preliminary tests were carried out to use P-NMR in future experiments to assign these
residues.
The second part of this thesis aimed to a structural characterisation of the receptor protein by
X-ray crystallography. For the first time, it was possible to obtain crystals of the full-length
ethylene receptor AtETR1. The crystals diffracted to a resolution of about 11 to 12 Å, but data
could not be used for structure determination due to the high mosaicity and anisotropic spot
distribution. Comprehensive optimisation including protein modification and variations of the
experimental setup was tried but did not improve diffraction quality.
For that reason the crystallisation study was expanded to orthologous ETR1s from the moss
Physcomitrella patens subsp. patens and the tomato Lycopersicon esculentum. Expression and
purification protocols were established for both proteins, and first crystallisation screens already
gave spherulitic crystals. Although these crystalline structures are not suitable for diffraction
analysis, they outline starting conditions for future crystallisation approaches.

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