Membrane binding properties of Disabled-2

........................................................................................................................... ii Dedication ........................................................................................................................ iii Acknowledgment ............................................................................................................. iv Abbreviations .................................................................................................................... v Table of content .............................................................................................................. vii List of tables ..................................................................................................................... ix List of figures .................................................................................................................... x CHAPTER 1: INTRODUCTION ....................................................................................... 1 1.1 Literature review ................................................................................................... 1 1.1.1 Background ...................................................................................................... 1 1.1.2 The role of Dab2 in endocytosis ...................................................................... 3 1.1.3 The role of Dab2 in hemostasis ....................................................................... 4 1.1.4 N-PTB region ................................................................................................... 6 1.2 Hypothesis ............................................................................................................. 8 1.3 Objectives .............................................................................................................. 9 1.3.1 Specific objectives: .......................................................................................... 9 1.4 Significance ......................................................................................................... 12 CHAPTER 2: MATERIALS AND METHODS .............................................................. 13 2.1 Materials .............................................................................................................. 13 2.2 DNA cloning, plasmids, and protein expression and purification ...................... 13 2.3 Sample preparation for Circular Dichroism and Tryptophan Fluorescence spectroscopy studies ........................................................................................................ 14 2.4 Circular Dichroism Spectroscopy ....................................................................... 15 2.5 Tryptophan fluorescence spectroscopy ............................................................... 16 2.6 Acrylamide quenching ........................................................................................ 18 2.7 NMR spectroscopy .............................................................................................. 20 2.8 Liposome binding assay ...................................................................................... 22 2.9 Statistical analysis ............................................................................................... 23 CHAPTER 3: RESULTS AND DISCUSSION ................................................................ 24 3.1 Structural properties of Dab2 N-PTB .................................................................. 24 3.2 Dab2 N-PTB is relatively stable in DDM micelles ............................................. 28 3.3 Sulfatide binding induces structural changes in Dab2 N-PTB ............................ 34 3.4 PIP2 binding stabilizes Dab2 N-PTB without causing any structural changes in the protein ....................................................................................................................... 40 viii 3.5 Role of Trp94 in Dab2 membrane interactions ................................................... 44 3.6 Sulfatides, but not PIP2, is involved in membrane penetration of Dab2 ............ 48 CHAPTER 4: SUMMARY AND CONCLUSIONS ........................................................ 54 REFERENCES: ................................................................................................................ 57