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    Progress towards Co-crystalization of the E. coli Membrane Protein Intimin with Engineered Peptide-specific Antibody Fragments

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    HEANER-UNDERGRADUATERESEARCHOPTIONTHESIS-2015.pdf (2.672Mb)
    Date
    2016-01-25
    Author
    Heaner, David Prince
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    Abstract
    The determination of membrane protein structures is critical for the development of new pharmaceutical agents. Conventionally, membrane proteins are solubilized by the use of mild detergents. However, due to the lack of hydrophilic residues available to make crystal contacts and interference by the large detergent micelle, the quality of diffraction and resolution level needed for de novo structure determination is usually not obtained. In addition to the conventional detergent method, a new method using engineered single chain antibody fragments (scFv) and a Fab antibody fragment have been developed for use as crystallization chaperones. The scFv and Fab fragment interact with the membrane protein of interest via the EYMPME (EE) tag, which is selectively mutated into a hydrophilic loop of the protein. The membrane protein-antibody fragment complex may enter crystallization trials with the antibody fragment driving the complex nucleation through the formation of numerous, strong crystal contacts. Such a co-crystallization method with anti-EE scFv and Fab fragments provides the protein crystallographer with a “crystallization toolbox” that can be used for any crystallographic study of a protein of interest. Through size exclusion chromatography and SDS-PAGE analysis, complexation of the β-barrel membrane protein intimin harboring the EE tag with scFv/EE and Fab/EE has been shown to occur, from which crystallization trials have ensued. Formation of a co-crystal has proven to be difficult, which can be explained in part through molecular dynamics simulations of the mutated intimin L4 loop. This thesis work will present results and conclusions for this novel co-crystallization method.
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    http://hdl.handle.net/1853/54496
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    • Undergraduate Research Option Theses [631]
    • School of Chemistry and Biochemistry Undergraduate Research Option Theses [46]

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