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    Conformational Analysis of Disordered Proteins Using H/D Exchange Mass Spectrometry

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    Keppel_ku_0099D_12598_DATA_1.pdf (2.328Mb)
    Issue Date
    2013-05-31
    Author
    Keppel, Theodore Robert
    Publisher
    University of Kansas
    Format
    182 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Chemistry
    Rights
    This item is protected by copyright and unless otherwise specified the copyright of this thesis/dissertation is held by the author.
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    Abstract
    Intrinsically disordered proteins (IDPs) represent a growing area of scientific interest. The prevalence of IDPs within the proteomes of complex organisms and the importance of these proteins in cellular functions has become apparent. The application of hydrogen/deuterium exchange mass spectrometry (H/D-MS) to study IDPs is presented in this dissertation. Results that demonstrate improvements to the H/D-MS method are also presented. A thermoelectrically cooled refrigeration system was developed in order to house LC components necessary for bottom-up H/D-MS. This system was used to keep solvent temperatures low and stable, ensuring reproducible and minimized back exchange. Two model IDPs, the interacting domains of CREB binding protein (CBP, residues 2059-2117) and activator of thyroid and retinoid receptors (ACTR, residues 1018-1088), were analyzed using H/D-MS. CBP and ACTR represent two classes of IDPs: the molten globule and random coil, respectively. The mutual synergistic folding phenomenon observed when a complex is formed between these two proteins was also analyzed. A lower limit of several seconds of D2O exposure time is imposed when manual pipetting is used to add label and quench buffers to protein samples. A quench flow apparatus was used to extend the lower time scale limit of exposure times to 42 milliseconds. When CBP and ACTR were labeled using the quench flow apparatus, analyses revealed subtle exchange protection in specific regions of both proteins. An H/D-MS study of the conformational dynamics of β-glucuronidase was also conducted. The destabilization of β-glucuronidase that results from the W492G mutation and the partial conformational rescue that results from indole addition were analyzed. A new method to interpret and normalize H/D-MS data was introduced to improve intra- and inter-laboratory comparability.
    URI
    http://hdl.handle.net/1808/12331
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    • Chemistry Dissertations and Theses [336]
    • Dissertations [4474]

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    785-864-8983
    KU Libraries
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    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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