Title page for ETD etd-05032011-071428


Type of Document Master's Thesis
Author Alajlouni, Ruba
URN etd-05032011-071428
Title Membrane binding properties of Disabled-2
Degree Master of Science
Department Biological Sciences
Advisory Committee
Advisor Name Title
Capelluto, Daniel G. S. Committee Chair
Lawrence, Christopher B. Committee Member
Sible, Jill C. Committee Member
Keywords
  • 5-bisphosphate
  • Circular Dichroism
  • Micelles
  • Sulfatide
  • Tryptophan Fluorescence
  • Disabled-2
  • Nuclear Magnetic Resonance
  • Phosphatidylinositol 4
  • Acrylamide quenching
Date of Defense 2011-04-21
Availability unrestricted
Abstract
Disabled-2 (Dab2) is an adapter protein that interacts with cell membranes and it is involved in several biological processes including endocytosis and platelet aggregation. During endocytosis, the Dab2 phosphotyrosine-binding (PTB) domain mediates protein binding to phosphatidylinositol 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane and helps co-localization with clathrin coats. Dab2, released from platelet alpha granules, inhibits platelet aggregation by binding to the αIIbβ integrin receptor on the platelet surface through an Arg-Gly-Asp (RGD) motif located within the PTB domain. Alternatively, Dab2 binds sulfatides on the platelets surface, and this binding partition Dab2 in two pools (sulfatide and integrin receptor-bound states), but the biological consequences of lipid binding remain unclear. Dab2 binds sulfatides through two basic motifs located on its N-terminal region including the PTB domain (N-PTB). We have characterized the binding of Dab2 to micelles, which are widely used to mimic biological membranes. These micellar interactions were studied in the absence and presence of Dab2 lipid ligands, sulfatides and PIP2. By applying multiple biochemical, biophysical, and structural techniques, we found that whereas Dab2 N-PTB binding to PIP2 stabilized the protein but did not contribute to the penetration of the protein into micelles, sulfatides induced conformational changes and facilitated penetration of Dab2 N-PTB into micelles. This is in agreement with previous observation that sulfatides, but not PIP2, protect Dab2 N-PTB from thrombin cleavage. By studying the mechanism by which Dab2 targets membranes, we will have the opportunity to manipulate its function in different lipid-dependent biological processes.
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