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    Understanding Structural Stability of Pharmaceutically Relevant Macromolecular Complexes: A Biophysical and Biochemical Approach

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    Esfandiary_ku_0099D_10532_DATA_1.pdf (17.65Mb)
    Issue Date
    2009-08-10
    Author
    Esfandiary, Reza
    Publisher
    University of Kansas
    Format
    247 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Pharmaceutical 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
    Macromolecules, due to their large size and complexity are prone to a variety of physical and chemical degradations. Development of stable formulations that retain the macromolecule's stability and activity over its designated shelf life is therefore a crucial step in the production of such complexes as safe and effective therapeutics. One rapid and systematic formulation tool is the utility of the empirical phase diagrams (EPDs) which have enhanced our understanding of the response of the structure and stability of proteins to environmental perturbations. In the case of more complex macromolecular systems (e.g., multi-domain fusion proteins, large recombinant proteins, and viruses), however, the measured stability is presumably the sum of all components. This makes interpretations at the molecular level difficult. Herein, we have investigated the structural stability of three macromolecular systems of varying complexity to see if their structural stability can at least be partially understood in terms of the behavior of their individual domains and components. We have examined the effect of the observed structural alterations on the losses in activity. We have discussed how this information can be used in designing high throughput screening assays for identification of solution stabilizers for development of optimal formulations. The utility of the obtained information in interpretation of the biological functions of these systems in vivo is also evaluated. Empirical Phase Diagrams constructed based on techniques sensitive to transitions due to alterations of protein motions have been shown to provide information above and beyond that obtained by the static approach. Therefore, integration of techniques that detect extensive and subtle conformational alterations of proteins, as well as structural fluctuations into an EPD appears to be crucial. Herein, we show that plots of the temperature dependent 2nd derivative peak positions of aromatic residues have measurable slopes prior to protein unfolding and that these slopes are sensitive to the dielectric properties of the surrounding microenvironment. We further demonstrate that these slopes correlate with hydration of the buried aromatic residues in protein cores and can therefore be used as qualitative probes of protein dynamics.
    URI
    http://hdl.handle.net/1808/6180
    Collections
    • Dissertations [4473]
    • Pharmaceutical Chemistry Dissertations and Theses [141]

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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
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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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