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    Contribution of Ordered Water Molecules and a Crucial Phenylalanine to Cooperative Pathway(s) in Scapharca Dimeric Hemoglobin: a Dissertation

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    Authors
    Pardanani, Animesh Dev
    Faculty Advisor
    Dr. William Royer, Jr.
    Academic Program
    Biochemistry and Molecular Pharmacology
    UMass Chan Affiliations
    Biochemistry and Molecular Pharmacology
    Document Type
    Doctoral Dissertation
    Publication Date
    1997-06-01
    Keywords
    Hemoglobins
    Blood Chemical Analysis
    Amino Acids, Peptides, and Proteins
    Animal Experimentation and Research
    Fluids and Secretions
    Inorganic Chemicals
    
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    Abstract
    The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis binds oxygen cooperatively and thus offers a simple model system for studying communication between two chemically identical sites. Although the individual subunits of HbI have the same myoglobin-fold as mammalian hemoglobins, the quaternary assemblage is radically different. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins. Analysis of structures of this hemoglobin in the liganded (02or CO) and unliganded states has provided a framework for understanding the role of individual amino acid side-chains in mediating cooperativity. The work presented in this dissertation has directly tested the central tenets of the proposed structural mechanism for cooperativity in HbI, illuminating the key roles played by residue Phe 97 and interface water molecules in intersubunit communication. Heterologous expression of Scapharca dimeric hemoglobin: A synthetic gene has been utilized to express recombinant RbI in Escherichia coli. The HbI apoprotein constitutes 5-10% of the total bacterial protein in this system. Addition of the heme precursor δ-aminolevulinic acid to the expression culture results in a ~3-fold increase in the production of soluble hemoglobin. Recombinant HbI has been successfully purified to homogeneity, resulting in a final yield of 80-100 mg of pure holoprotein from a 12 L expression culture. Analysis of recombinant HbI reveals its oxygen binding properties to be indistinguishable from native HbI. It was necessary to correct a protein sequence error by mutating residue Asn 56 to aspartate in order to obtain diffraction quality crystals, that are isomorphous to native HbI crystals. These recombinant HbI crystals diffract to high resolution, permitting the functional effects of mutant HbI proteins to be correlated with detailed structural analysis.
    DOI
    10.13028/jtap-3c76
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/31636
    Rights
    Copyright is held by the author, with all rights reserved.
    ae974a485f413a2113503eed53cd6c53
    10.13028/jtap-3c76
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    Morningside Graduate School of Biomedical Sciences Dissertations and Theses

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