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Conformational Changes in Ezrin Analyzed by Förster Resonance Energy Transfer

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Conformational Changes in Ezrin Analyzed by Förster Resonance Energy Transfer Conformational changes in ezrin analyzed by Förster resonance energy transfer Dissertation am Fachbereich Biologie der Universität Münster Victoria Shabardina 2015 Biologie Conformational changes in ezrin analyzed by Förster resonance energy transfer Inaugural-Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften im Fachbereich Biologie der Mathematisch-Naturwissenschaftlichen Fakultät der Westfälischen Wilhelms-Universität Münster vorgelegt von Victoria Shabardina Aus Kirov 2015 Dekan: Prof. Dr. Wolf-Michael Weber Erster Gutachter: Prof. Dr. Volker Gerke Zweiter Gutachter: Prof. Dr. Martin Bähler Tag der mündlichen Prüfung: 8.12.2015 Tag der Promotion: 18.12.2015 Contents Summary ......................................................................................................................................... V 1 Introduction .................................................................................................................................... 1 1.1 The ERM protein family ................................................................................................................ 1 1.2 ERM proteins and their conservative features ............................................................................ 1 1.3 Ezrin is involved in cell cortex organization and signaling cascades ............................................. 4 1.4 Dormant and activated states of ezrin.......................................................................................... 7 1.4.1 Role of PIP2 in the cell ............................................................................................................. 8 1.4.2 Protein phosphorylation as one of the main regulators of cell functioning ............................. 10 1.4.3 Structure and conformations of ezrin ..................................................................................... 11 1.5 Ezrin activation hypothesis ......................................................................................................... 16 1.6 Aim of the study and general experimental approach .............................................................. 20 2 Materials ....................................................................................................................................... 25 2.1 Chemicals and reagents .............................................................................................................. 25 2.2 Markers ....................................................................................................................................... 26 2.3 Kits ............................................................................................................................................... 26 2.4 Antibodies ................................................................................................................................... 27 2.5 Enzymes ..................................................................................................................................... 27 2.6 Cell lines ...................................................................................................................................... 27 2.7 Bacterial strains ........................................................................................................................... 28 2.8 DNA-constructs ........................................................................................................................... 28 2.8.1 Eukaryotic and bacterial expression vectors ........................................................................... 28 2.8.2 Oligonucleotides .................................................................................................................... 29 2.9 Equipment ................................................................................................................................... 29 2.10 Devices ...................................................................................................................................... 29 3 Methods ........................................................................................................................................ 31 3.1 Cell biology methods ................................................................................................................... 31 3.1.1 Cultivation of adherent eukaryotic cells .................................................................................. 31 3.1.2 Cryo-stocks ............................................................................................................................ 31 3.1.3 EGF stimulation experiments ................................................................................................ 31 3.1.4 Confocal immunofluorescence imaging.................................................................................. 31 3.1.5 Transient transfection ............................................................................................................ 32 3.1.6 Stable transfection ................................................................................................................. 33 I 3.2 Molecular biology methods ........................................................................................................ 33 3.2.1 Plasmids cloning .................................................................................................................... 33 3.2.2 Maxi-preps ............................................................................................................................. 36 3.3 Biochemical methods .................................................................................................................. 36 3.3.1 SDS-PAGE ........................................................................................................................... 36 3.3.2 Western Blot analysis ............................................................................................................. 37 3.3.3 DNA gel-electrophoresis ....................................................................................................... 38 3.3.4 Bacterial expression and purification of the proteins .............................................................. 39 3.3.5 Recombinant expression and purification of proteins from HEK293-T cells ......................... 40 3.3.6 Fluorescent labeling of proteins ............................................................................................. 41 3.3.7 Bradford assay ....................................................................................................................... 42 3.3.8 Liposomes preparation .......................................................................................................... 42 3.3.9 Liposomes pelleting assay ...................................................................................................... 42 3.3.10 Supported lipid bilayer preparation and protein binding ....................................................... 43 3.3.11 Reflectometric Interference Spectroscopy - RIfS .................................................................. 44 3.3.12 Intra- and inter-molecular FRET on supported lipid bilayers ............................................... 44 3.3.13 Enzymatic protein digest ...................................................................................................... 45 4 Results ................................................................................................................................................ 47 4.1 Expression and purification of the recombinant ezrin, ezrinT567D and their GFP-fused derivatives using the BL21 strain of E.coli and HEK293T cells .......................................................... 48 4.2 Liposome pelleting assay: the fluorescent derivatives of ezrin bind to PIP2 and DOGS-Ni-NTA containing liposomes like the wild type protein ............................................................................... 50 4.3 EzrGFP and DGFP are recruited to the cell membrane in EGF activated A431 cells similar to endogenous ezrin .............................................................................................................................. 51 4.4 DyLight405EzrGFP is not suitable for FRET analysis ................................................................... 55 4.5 Cy3EzrGFP shows FRET ............................................................................................................... 56 4.6 Reflectometric Interference Spectroscopy (RIfS) showes binding of the Cy3-labeled ezrin derivatives to PIP2- and DOGS-Ni-NTA-containing SSLB ................................................................... 59 4.7 Fluorescent spectra, FRET in solution ......................................................................................... 61 4.8 Analysis of FRET efficiency on solid supported bilayers ............................................................. 64 5 Discussion ...................................................................................................................................... 73 5.1 GFP-tag does not change properties of ezrin in the cells and in in vitro binding assays ............ 73 5.2 Properties of Cy3EzrGFP and DyLight405EzrGFP as FRET constructs ......................................... 75 5.3 PIP2 binding and threonine 567 phosphorylation contribute to conformational change in ezrin simultaneously .................................................................................................................................
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