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Characterization of Synaptotagmin 1 Function in Calcium Dependent Neuronal Exocytosis

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Characterization of Synaptotagmin 1 Function in Calcium Dependent Neuronal Exocytosis CHARACTERIZATION OF SYNAPTOTAGMIN 1 FUNCTION IN CALCIUM DEPENDENT NEURONAL EXOCYTOSIS Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität zu Göttingen vorgelegt von Anand Radhakrishnan aus Muar, Johor, Malaysia Göttingen, der 20. März 2007 Referent: Prof. Reinhard Jahn Korreferent: Prof. Ivo Feußner Tag der mündlichen Prüfung: 2. Mai 2007 ii Eidesstattliche Erklärung: Hiermit erkläre ich an Eides Statt, dass ich die vorliegende Arbeit: “Characterization of Synaptotagmin 1Function in Neuronal Exocytosis“ selbständig verfasst ohne unerlaubte Hilfe angefertigt habe. Göttingen, 20. März 2007 Anand Radhakrishnan iii For my parents… Acknowledgements The work presented here was only possible due to contributions from many people both in the lab as well as outside. I would like to thank Prof. Jahn for giving me the opportunity to participate in the Molecular Biology program and subsequently, allowing me to work in his lab for my masters and now, PhD. I am indebted to Dr. Dirk Fasshauer whose patient guidance allowed me to learn to use the tools I needed for this work. I am also grateful for the many hours of discussion and the constant encouragement that helped me continue my work over the years. A big thank you to Dr. Gottfried Mieskes, who’s ever-ready to lend a helping hand in everything from packing a column to discussions on the MPG senate. I am grateful to my lab mates and friends, Alexander, Pawel, Katrin, Mads, Nickias, Nathan, Sina and Ioanna for the wonderful time both in and out of the lab and whose constant encouragement during my thesis-writing period was indispensable in helping me finish. I am grateful to all other “Neurobiologie” group members that contributed to the great work environment these past years (it’s true that this is the “Disneyland of Science”). And finally, I would like to express my love and gratitude to the two most important people in my life, my parents, whose unflinching love and support have helped me reach this far. i Table of Content Acknowledgements ...............................................................................................................i Table of Content...................................................................................................................ii List of Figures & Tables......................................................................................................v Abbreviations ....................................................................................................................viii Abstract.................................................................................................................................1 Kurzfassung..........................................................................................................................3 1. Introduction......................................................................................................................5 1.1 Calcium-dependent exocytosis and the hunt for a neuronal calcium sensor ...............5 1.2 Genetic-based analysis of Synaptotagmin 1 ................................................................6 1.3 Domain Structure of Synaptotagmin 1 ........................................................................6 1.3.1 C2 domains ...........................................................................................................7 1.4 Calcium-dependent Phospholipid Binding ................................................................11 1.4.1 Membrane penetration and Cooperativity of Synaptotagmin C2 domains.........11 1.4.2 Effect of Phosphatidyl Inositol (4,5)bisphosphate (PIP2) on Synaptotagmin ....................................................................................................14 1.5 Interaction of Synaptotagmin with the SNARE complex..........................................15 1.5.1 Biochemical Characterization of Synaptotagmin - SNARE binding..................18 1.5.2 Complexin...........................................................................................................21 1.6 Mechanism of calcium dependent exocytosis: Integrated in vitro approaches .........23 1.7 Objective....................................................................................................................26 2. Methods...........................................................................................................................28 2.1 Plasmid Construction.................................................................................................28 2.2 Protein Expression and Purification ..........................................................................31 2.3 Protein labelling.........................................................................................................33 2.4 Lipid Mixes and Liposomes ......................................................................................33 2.5 Absorption Spectroscopy...........................................................................................34 2.6 SNARE ternary complex formation ..........................................................................35 2.7 Native PAGE .............................................................................................................36 2.8 Isothermal Titration Calorimetry (ITC).....................................................................37 2.9 Fluorescence Spectroscopy........................................................................................38 2.9.1 Interaction of Synaptotagmin with the lipid membrane .....................................38 ii 2.9.2 Fluorescence measurements of SNARE complex labelled with SNAP25 48IAANS ............................................................................................................39 2.9.3 Fluorescence Anisotropy ....................................................................................41 2.9.3.1 SNARE assembly kinetics...........................................................................41 2.9.3.2 Interaction of Complexin to the SNARE complex ......................................41 2.9.3.3 Binding of Synaptotagmin to the SNARE ternary complex (+TMD).........42 2.9.4 Interaction of Synaptotagmin to the SNARE complex: A FRET-based approach..............................................................................................................44 3. Results.............................................................................................................................46 3.1 Calcium binding of the Synaptotagmin C2 domains.................................................46 3.1.1 Calcium binding of the isolated Synaptotagmin C2 domains.............................47 3.1.2 Calcium binding of Synaptotagmin with the tandem C2 domains .....................49 3.2 Lipid Binding.............................................................................................................55 3.2.1 Influence of lipid composition on the binding of Synaptotagmin to the liposome membrane............................................................................................57 3.3 Synaptotagmin – SNARE Interaction........................................................................60 3.3.1 Binding of Synaptotagmin to the SNARE..........................................................60 3.3.1.1 Synaptotagmin – SNARE interaction: A gel-based approach.....................60 3.3.1.2 Synaptotagmin – SNARE interaction: A spectroscopic approach...............67 3.3.1.2.1 Interaction of Synaptotagmin with SNARE complex labelled with IAANS ................................................................................................67 3.3.2.1.2 Anisotropy measurements of Synaptotagmin – SNARE complex interaction ...........................................................................................71 3.3.2 Functional Characterization of Synaptotagmin-SNARE Interaction..................76 3.3.2.1 Influence of Synaptotagmin on the kinetics of SNARE complex assembly76 3.3.2.2 Synaptotagmin – SNARE complex interaction in liposome membranes....78 3.3.3 Synaptotagmin – SNARE interaction: A FRET-based approach .......................80 4. Discussion .......................................................................................................................89 4.1Calcium Binding.........................................................................................................89 4.1.1 Limitations of ITC ..............................................................................................89 4.1.2 Calcium Binding mechanism of the Synaptotagmin C2 domains ......................90 4.1.2.1 Enthalpy – Entropy Compensation..............................................................93 4.1.2.2 Low affinity calcium binding ......................................................................94 iii 4.2 Membrane Organization and Lateral Diffusion.........................................................96 4.2.1 Membrane microdomains and Synaptotagmin ...................................................97 4.3 Binding of Synaptotagmin to the SNARE complex ................................................100 4.3.1 Synaptotagmin – SNARE interaction: Binding Site.........................................101 4.3.2 Synaptotagmin – SNARE interaction: Functional Relevance ..........................102 5. Conclusion & Outlook.................................................................................................107 Reference
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