Bioanalytical Techniques to Investigate Acyl Carrier Protein-Protein Interactions /
- Author(s): Rothmann, Michael Isaac
- et al.
The acyl carrier protein (ACP) is a small, stable protein with exquisite specificity and reactivity. The work outlined in this thesis demonstrates the design of assays that leverage an understanding of the reactivity of ACP for the design of novel bioanalytical systems. The systems designed here rely on a variety of techniques including mass spectrometry, fluorescent microscopy, electrophoresis mobility and in gel fluorescent scanning, high performance liquid chromatography, and enzyme immobilization. This array of techniques allows the biochemist a host of opportunities not only to investigate the cellular role of ACP but also the possibilities to use this protein as a tool in the investigation of other biological systems. ACP plays a vital role in cellular metabolism and therefore an understanding of the cellular processes that involve ACP is crucial for understanding the overall metabolic processes of the cell. Much of the work presented here aims to elucidate the protein-protein interactions of ACP and the extent of its cellular interactome. Some enzyme interactions described here have been previously annotated for their roles and in closely associated processes and others are more unexpected, suggesting allosteric regulation and involvement in pathways that are not so closely related to the primary role of ACP in fatty acid biosynthesis. Also, fundamental questions about how ACP and its cofactors are synthesized and their levels regulated are posed and investigated. Finally, new techniques to aid the preparation of various forms of ACP are presented. Using the favorable properties of enzyme immobilization, we enable expedited ACP preparation for use in all of the applications mentioned above