Contribution of Ordered Water Molecules and a Crucial Phenylalanine to Cooperative Pathway(s) in Scapharca Dimeric Hemoglobin: a Dissertation
Authors
Pardanani, Animesh DevFaculty Advisor
Dr. William Royer, Jr.Academic Program
Biochemistry and Molecular PharmacologyUMass Chan Affiliations
Biochemistry and Molecular PharmacologyDocument Type
Doctoral DissertationPublication Date
1997-06-01Keywords
HemoglobinsBlood Chemical Analysis
Amino Acids, Peptides, and Proteins
Animal Experimentation and Research
Fluids and Secretions
Inorganic Chemicals
Metadata
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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-3c76Permanent Link to this Item
http://hdl.handle.net/20.500.14038/31636Rights
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10.13028/jtap-3c76