MeagherĬrystallization and preliminary X-ray analysis of Na-ASP-1, a multi-domain pathogenesis-related-1 protein from the human hookworm parasite Necator americanus, Dr.Mehmet Inan, Oluwatoyin A. Smith, Robert Webb, Bonnie Loveless, Ian Henderson, and Michael M. Dux, Rick Barent, Jayanta Sinha, Mark Gouthro, Todd Swanson, Ardis Barthuli, Mehmet Inan, John T. Purification and scale-up of a recombinant heavy chain fragment C of botulinum neurotoxin serotype E in Pichia pastoris GS115, Michael P. MeagherĭNA Polymerase Chain Reaction: A Model of Error Frequencies and Extension Rates, Hendrik J. Smith, Theresa Smith, Ian Henderson, John Ross, and Michael M. Taken together, this thesis work provides valuable insight into the means by which the CH domain of MICAL modulates its activity.Cell bank characterization and fermentation optimization for production of recombinant heavy chain C-terminal fragment of botulinum neurotoxin serotype E (rBoNTE(H c): Antigen E) by Pichia pastoris, Jayanta Sinha, Mehmet Inan, Sarah Fanders, Shinichi Taoka, Mark Gouthro, Todd Swanson, Rick Barent, Ardis Barthuli, Bonnie M. Finally, molecular dynamics simulations using this model revealed that a direct oxidation of actin’s Met44 by MICAL is possible, by an extension of the loop containing Met44 of actin. The structure of MICALFD-CH was used to build a possible model of the interaction of MICAL with actin. Kinetic studies reveal that the presence of the CH domain is important for substrate specificity.

Furthermore, the FD and CH domains are connected by a disordered linker, which could be important for accommodating the binding of the FD to actin. The crystal structure of MICALFD-CH, determined to 2.9-Å resolution, reveals that the CH domain does not interact with the active site in the FD. In this thesis work, the modulation of MICAL activity by the CH domain was characterized structurally, biochemically, and thermodynamically.

However, modulation of MICAL activities by its non-redox CH domain is poorly understood. CH domains are typically found in actin-binding proteins. Studies showed that MICALFD and MICALFD-CH (a construct containing the FD and CH domains only) can bind and oxidize Met44 on actin filaments, thereby affecting their polymerization dynamics. Our laboratory has previously determined the crystal structure of MICALs FD (MICALFD) and showed that it uses NADPH as the reductant. Proper axon guidance is vital in neural development processes such as neuronal cell-migration, axonal branching, path finding, and fasiculation/defasiculation. Axon guidance is the process by which growing axons respond to extracellular cues that guide them towards their appropriate targets. In axon guidance, MICAL is a key molecule that links the extracellular signal from semaphorins –a class of repulsive guidance cues– to the reorganization of the cytoskeleton. MICAL (Molecule Interacting with CasL) is a 1048 amino acid protein consisting of a flavin-containing monooxygenase domain (FD) with redox activity, a Calponin homology (CH) domain, a LIM (Lin-11, Isl-1, Mec-3) domain, a proline-rich region, and a C-term region containing coiled-coil ERM α-like domain.

Mical biochemical