Protein-Protein Interactions in Natural Product Biosynthesis

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Project Summary This project aims to develop a new synthetic tool to investigate the ligand·protein and protein·protein interactions in carrier protein-dependent biosynthetic pathways including fatty acid synthase (FAS), polyketide synthase (PKS), and non-ribosomal peptide synthetase pathways (NRPS). The natural products produced from these pathways constitute a majority of therapeutics ranging from antibiotics and antifungals to anticancer compounds. Manipulating and controlling the biosynthesis of these pathways toward the production of new bioactive compounds has been a long-term goal of the field. Developing an understanding of the protein·protein interactions will allow us to make strides toward these long-term goals, as interactions between the carrier protein and partner protein domains guide the processivity and reactivity of the pathway. In the Burkart lab, we have previously developed chemical biology tools to study snapshots of these interactions or visualize them through the use of fluorescent dyes. It is proposed in this project to create a new site-specific 15N isotopically labeled tool that will be able to visualize changes in the environment of the ligand attached to the carrier protein and give information about chain flipping of this prosthetic arm through 15N nuclear magnetic resonance (NMR) studies. This tool will be able to mimic the native prosthetic arm while giving real time feedback on the temporal location of the probe and its structural surroundings in a simplified form. This versatile tool will be able to give information on ligand·protein interactions and chain flipping phenomena in multiple systems through its adaptable synthetic route including Escherichia coli AcpP with the chain-length specific LipB, and with the functional group specific ketoreductase and enoyl reductase E. coli FAS domains.