The dimerization of parotid secretory protein

......................................................................................................... iv LIST OF TABLES .............................................................................................. viii LIST OF FIGURES .............................................................................................. ix INTRODUCTION ................................................................................................. 1 MATERIALS AND METHODS ........................................................................ .11 Clone Production .................................................................................... 11 Purification of Fusion Protein .................................................................. 17 Protein Assay ........................................................................................... 17 Isolation of Parotid Secretory Granules ................................................... 18 Crosslinking ............................................................................................ 18 Affinity Binding Protocol ........................................................................ 19 Computer Modeling of Quaternary Structure .......................................... 20 Protein-Lipid Binding .............................................................................. 20 RESULTS ............................................................................................................ 22 Crosslinking with Formaldehyde/ Bst 3 .................................................... 22 Affinity Binding Experiment .................................................................. 25 Buffer Optimization ................................................................................. 31 vii Chlorampenicol Acetlytransferase (CAT) Control Experiment ............. 34 Human PSP Experiments ........................................................................ 35 Titration of reticulocyte lysate to resin .................................................... 37 I-Tasser Computer modeling .................................................................. 42 Symmdock Dimerization Modeling ........................................................ 46 Rosetta Software 3-D Modeling .............................................................. 51 Testing Stacked Dimer Model with Mutants ........................................... 55 Testing Dimer Model #2 (End-to-End model) ........................................ 59 Lipid Binding ........................................................................................... 62 DISCUSSION ...................................................................................................... 67 REFERENCES .................................................................................................... 72 CURRICULUM VITAE ...................................................................................... 76