Protein Complexes in Neurodegenerative Diseases

......................................................................................................................................... I DECLARATION ............................................................................................................................... III ACKNOWLEDGEMENTS ............................................................................................................. IV FIGURES ............................................................................................................................................. V TABLES .............................................................................................................................................. VI ABBREVIATIONS ......................................................................................................................... VII TABLE OF CONTENTS .................................................................................................................. IX 14-3-3 PROTEINS, LIPID RAFTS AND NEURODEGENERATIVE DISEASES ................... 1 1.1. 14-3-3 Proteins .................................................................................................................... 2 1.1.1. Identification and Characterisation of 14-3-3 Proteins ................................................ 2 1.1.2. The Crystal Structure of 14-3-3 ....................................................................................... 5 1.1.3. 14-3-3 Isoforms .................................................................................................................. 9 1.1.4. 14-3-3 and Neurodegenerative Diseases ...................................................................... 18 1.2. Lipid Rafts ......................................................................................................................... 29 1.2.1. What are Lipid Rafts? ..................................................................................................... 29 1.2.2. Lipid Raft Composition .................................................................................................. 30 1.2.3. Lipid Rafts and Neurodegenerative Diseases ............................................................. 34 1.3. Spinocerebellar Ataxia Type 1 (SCA1) .......................................................................... 39 1.3.1. Clinical Effects of SCA1.................................................................................................. 39 1.3.2. Disease Pathology ........................................................................................................... 41 1.3.3. Ataxin-1 and 14-3-3 ......................................................................................................... 48 1.4. Aims of Project ................................................................................................................. 53 MATERIALS AND METHODS .................................................................................................... 55 2.1. Molecular Biology ............................................................................................................ 55 2.1.1. Agarose Gel Electrophoresis ......................................................................................... 56 2.1.2. Primer Design .................................................................................................................. 56 2.1.3. Polymerase Chain Reaction (PCR) with Pfu Polymerase .......................................... 57 2.1.4. Restriction Digest ............................................................................................................ 58 2.1.5. Gel Extraction .................................................................................................................. 59 2.1.6. Ligation of Insert DNA into Vector DNA.................................................................... 59 2.1.7. Preparation of Competent Bacteria .............................................................................. 60 2.1.8. Transformation of Bacteria ............................................................................................ 60 2.1.9. Production of Glycerol Stocks ....................................................................................... 61 2.1.10. Purification of Plasmid DNA ...................................................................................... 61 2.1.11. Quantification of Plasmid DNA.................................................................................. 61 2.2. Expression and Purification Techniques ...................................................................... 62 2.2.1. Expression of Recombinant Proteins ............................................................................ 63 2.2.2. Immobilised Metal Affinity Chromatography (IMAC) by Gravity Flow ............... 64 2.2.3. Ion-Exchange Chromatography .................................................................................... 68 2.2.4. Gel Filtration Chromatography .................................................................................... 68 2.2.5. Concentration and Storage of Proteins ........................................................................ 69