Wiess School of Natural Sciences
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Biochemistry & Cell Biology
Earth Science
Ecology & Evolutionary Biology
Physics & Astronomy

Expression, Folding, and Assembly Mechanisms of Monomeric Myoglobins and Oligomeric Hemoglobins

Thesis Defense

Graduate and Postdoctoral Studies

By: Premila Samuel Mohan Dass
Doctoral Candidate
When: Friday, October 13, 2017
2:00 PM - 4:00 PM
Where: Keck Hall
Abstract: Despite differing in quaternary structure and protein sequence, mammalian myoglobins and hemoglobins share similar overall globin folds and nearly identical active site structures. The folding mechanism for monomeric apomyoglobin is a well-characterized 2-step pathway involving a molten globule intermediate containing an unfolded heme pocket. Holomyoglobin assembly involves reversible hemin binding to both the molten globule and fully folded apomyoglobin. A wheat germ based cell-free expression assay was developed to show that production levels of folded holomyoglobins correlate quantitatively with their overall apomyoglobin stability constants. Higher cell-free expression levels were also observed for myoglobin mutants with heme cavity filling mutations that significantly increase apomyoglobin stability at the expense of hemin binding affinity. The new in vitro results are consistent with previous observations of myoglobin expression in animal muscle cells and E. coli, all of which demonstrate that apomyoglobin stability is the key determinant of holoprotein expression. In contrast to myoglobin, the individual ? and ? apoglobin subunits of adult human hemoglobin A (HbA) are extremely unstable, despite being structurally similar to apomyoglobin. GdnHCl induced unfolding curves were measured for human apo- and holo- HbA, fetal hemoglobin, and recombinant hemoglobins with either heme cavity filling apolar mutations or a genetically crosslinked di-? subunit. A mathematical model for hemoglobin tetramer assembly was developed, starting with the mechanism for apohemoglobin folding and adding heme binding steps for each of the different apoprotein states. The unfolding pathway for the heterodimeric apohemoglobin is a 4-step, 5-state mechanism. The first step involves unfolding of the heme pockets to form a heterodimeric molten globule intermediate. This intermediate dissociates into mostly unfolded monomers that then either interact transiently or undergo complete unfolding. Reversible hemin binding to the folded ?? apohemoglobin dimer facilitates formation of the tetrameric ?1?2 interfaces, promoting the final assembly of the HbA tetramer. Both the experimental studies and mathematical modeling of hemoglobin assembly provide the framework for understanding human hemoglobinopathies arising from globin misfolding and for enhancing the production yields of heme proteins in bacterial and eukaryotic expression systems.