ANALYZING THE ROLE OF COVALENT MODIFICATIONS TO AN ARYL CARRIER PROTEIN IN DIRECTING SEQUENTIAL INTERACTIONS IN YERSINIABACTIN SYNTHETASE
| dc.contributor.advisor | Townsend, Craig A. | |
| dc.contributor.committeeMember | Frueh, Dominique P. | |
| dc.contributor.committeeMember | Tolman, Joel R. | |
| dc.contributor.committeeMember | Stivers, James T. | |
| dc.creator | Goodrich, Andrew Christopher | |
| dc.creator.orcid | 0000-0001-7799-7134 | |
| dc.date.accessioned | 2017-04-19T12:24:12Z | |
| dc.date.available | 2017-04-19T12:24:12Z | |
| dc.date.created | 2016-12 | |
| dc.date.issued | 2016-06-08 | |
| dc.date.submitted | December 2016 | |
| dc.date.updated | 2017-04-19T12:24:12Z | |
| dc.description.abstract | Nonribosomal peptide synthetases (NRPSs) are enzymatic systems found in bacteria and fungi responsible for the production of complex secondary metabolites. NRPSs are able to generate a remarkably diverse array of natural products from simple starting materials. This is due to their modular nature and assembly-line organization in which the growing product is passed from module to module with an individual monomer incorporated at each step. The modular nature of NRPSs makes them an attractive tool for synthetic biologists to generate novel natural products with desirable pharmaceutical or industrial properties. In principle, existing modules could be rearranged in a combinatorial fashion to produce an enormous number of new products. However, in practice this has not been successful, likely due to a lack of understanding of the mechanisms underlying NRPS synthesis. Each module is made up of at least three domains whose combined function leads to the selection, activation, and incorporation of a small molecule into the growing product. Domains called carrier proteins are first modified from an inactive apo form to an active holo form in which a 4’-phosphopantetheine (PP) moiety is attached to a conserved serine. Adenylation domains then select and activate a small molecule using ATP via formation of an acyl-adenylate and then load the small molecule onto a holo-carrier protein via formation of a thioester with the terminal thiol of the PP arm. Condensation domains then catalyze amide bond formation between substrates loaded on adjacent carrier proteins. During synthesis, individual domains must move relative to one another. Domains are also subject to both small and large-scale conformation changes. NRPS synthesis is therefore a complex process involving the interplay of catalysis, covalent modification, and conformational rearrangements. Understanding how these processes are orchestrated to achieve efficient synthesis will be necessary for rationally redesigning these systems. Here, I present my work aimed at dissecting the role of covalent modifications to carrier proteins in altering their structure and how this, in turn, modulates interactions with adenylation domains. In order to study the structure of a carrier protein in all of its forms, we first developed a novel method to characterize the loaded form by nuclear magnetic resonance (NMR) spectroscopy. We then solved the solution structures of a CP in all of its forms and characterized the NMR dynamics of the holo and substrate-loaded forms. Finally, we characterized the interaction between a carrier protein and an adenylation domain by fluorescence anisotropy, isothermal titration calorimetry, and NMR titration. Our results show that covalent modifications alter the structure and dynamics of the carrier protein and prosthetic moieties in a way that provides directionality to the interaction with the adenylation domain that parallels the chemical steps of elongation and thus promotes efficient synthesis. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://jhir.library.jhu.edu/handle/1774.2/40289 | |
| dc.language | en | |
| dc.publisher | Johns Hopkins University | |
| dc.publisher.country | USA | |
| dc.subject | Nonribosomal peptide synthetase | |
| dc.subject | carrier proteins | |
| dc.subject | substrate loading | |
| dc.subject | NMR | |
| dc.subject | protein-protein interactions | |
| dc.title | ANALYZING THE ROLE OF COVALENT MODIFICATIONS TO AN ARYL CARRIER PROTEIN IN DIRECTING SEQUENTIAL INTERACTIONS IN YERSINIABACTIN SYNTHETASE | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Biophysics and Biophysical Chemistry | |
| thesis.degree.discipline | Biophysics | |
| thesis.degree.grantor | Johns Hopkins University | |
| thesis.degree.grantor | School of Medicine | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Ph.D. |