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    SMALL MOLECULE BINDING OF INTRINSICALY DISORDERED PROTEINS: MULTIPLE BINDERS ON MULTIPLE SITES

    Cover for SMALL MOLECULE BINDING OF INTRINSICALY DISORDERED PROTEINS: MULTIPLE BINDERS ON MULTIPLE SITES
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    View/Open: Hammoudeh_georgetown_0076D_10346.pdf (21.MB) Bookview

    Creator
    Hammoudeh, Dalia
    Advisor
    Metallo, Steven J
    Abstract
    Intrinsically disordered (ID) proteins lack a stable tertiary structure and exist in an ensemble of rapidly inter-converting conformations. They are prevalent in human and other genomes, and are biologically active in their ID state. ID proteins are overrepresented in signaling and regulation, and are implicated in many diseases thus making them attractive targets for drug discovery. However, they are not considered druggable targets.
     
    This dissertation describes the finding of multiple discrete sites within the ID proteins c-Myc and Id2 which can selectively and independently bind to structurally diverse small organic molecules. These proteins are disordered in their monomeric state and only upon dimerization with a partner protein do they form a stable tertiary structure. The binding sites are composed of short contiguous stretches of amino acids, contain residues found to be conserved across the family of proteins but not in the targeted sequence, and are found in regions predicted to have low disorder probability. Four sites were identified in the 85 amino acid bHLH region of the oncoprotein c Myc using mutagenesis, fluorescence polarization, circular dichoism (CD), and nuclear magnetic resonance spectroscopy (NMR). Later, a relatively quick and simple strategy was used to identify three small molecule binding sites on the 41 amino acid HLH domain of Id2. This method entails screening short overlapping peptides by monitoring changes in their secondary structures via CD, and can be applied to any ID sequence. Structural studies of the ensemble averaged conformations of the peptides encompassing the binding sites before and after binding to the small molecules were performed using NMR. Peptide-small molecule complexes, although more rigid than the free peptides, are highly flexible. The small molecule inhibitors bind to the ID monomer proteins, affecting their structure at a local level only, preserving the overall disorder and preventing dimerization from taking place. We were able to successfully predict where binding sites would be found on the ID sequence of Id2.
     
    We have found that ID sequences contain many sites capable of specific binding to small organic molecules and propose a new strategy for identifying specific, high affinity small molecules binders of ID proteins.
     
    Description
    Ph.D.
    Permanent Link
    http://hdl.handle.net/10822/557936
    Date Published
    2009
    Subject
    c-Myc; Inhibiting protein-protein interaction; Intrinsically disordered protein; Intrinsically unstructured proteins; Protein-protein interaction; Small molecule inhibitors; Biophysics; Biochemistry; Biophysics, General; Chemistry, Biochemistry;
    Type
    thesis
    Embargo Lift Date
    2015-05-15
    Publisher
    Georgetown University
    Extent
    316 leaves
    Collections
    • Graduate Theses and Dissertations - Chemistry
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    Georgetown University Seal
    ©2009 - 2022 Georgetown University Library
    37th & O Streets NW
    Washington DC 20057-1174
    202.687.7385
    digitalscholarship@georgetown.edu
    Accessibility