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Structural and Biophysical Studies of Carrier Proteins and Their Cargo


Many natural products, including fatty acids, polyketides, and non-ribosomal peptides have pharmacologically valuable properties. These products are biosynthesized using diverse pathways, but they employ very similar machineries and chemical transformations. Central to these pathways are carrier proteins, which covalently tether a growing intermediate. Small, dynamic carrier proteins facilitate transport of intermediates to subsequent reaction partners within the biosynthetic pathway, and protect them from unfavorable reactions in the cytosol. This dissertation focuses on these carrier proteins, characterizing them both structurally and biophysically. Solution state protein NMR methods are ideal for studying carrier proteins, given their small size and high flexibility.

Several studies of the Escherichia coli fatty acid synthase (FAS) pathway yielded significant information about the structure and dynamics of the acyl carrier protein (ACP). Increasing the basic understanding of how modular synthases function, interact, and maintain product specificity has been a driving goal; such information is critical to engineering de novo modular pathways. Identification of the proteins in the pathways is essential, but understanding how they move and interact remains an intellectual bottleneck. Using mechanism-based crosslinking, the E. coli ACP was trapped in functional interaction with the cognate dehydratase protein, FabA. Structures were determined crystallographically in collaboration with the Tsai lab at the University of California, Irvine and by NMR in collaboration with the Opella lab at the University of California, San Diego. Residual Dipolar Couplings from NMR, together with Accelerated Molecular Dynamics studies in collaboration with the McCammon lab at the University of California, San Diego led to the identification of key residues necessary for this protein-protein interaction, and quantification of the ACP's dynamics both as a free protein and during interaction with a partner.

Furthermore, studies of the actinorhodin polyketide synthase (PKS) pathway led to insights into the sequestration and cyclization of polyketide intermediates. To prevent unwanted side-reactions of polyketide intermediates, geometrically appropriate atom replacement mimetic probes were synthesized and loaded onto the actinorhodin ACP. Chemical shift perturbations of the protein amide resonances allowed monitoring of sequestration, as probes of longer length demonstrated larger perturbation of more residues than shorter polyketide probes.

Additionally, studies of the prodigiosin and pyoluteorin pathways give new information about sequestration. Traditional NOE methods were employed to solve the solution NMR structure of the pyoluteorin carrier protein, PltL, in multiple cargo states. For the first time, sequestration of intermediates was observed in a non-ribosomal peptide synthetase (NRPS) system. Further studies are underway to determine if sequestration is observed in other NRPS systems.

Finally, a follow-up to the ACP FabA project is in an early manuscript stage, detailing the E. coli ACP interaction with another partner, the ketosynthase FabB. NMR titration experiments, demonstrating key residues in the ACP FabB interaction are reported. In vivo complementation of the wt ACP with mutated ACPs showed that perturbing the ACP FabB interaction reduces E. coli's ability to produce unsaturated fatty acids at lower temperatures, connecting in vivo observations to in vitro effects.

While challenges and unknowns remain, the work of this dissertation provides significant progress towards the possibilities of engineering novel pathways to produce pharmaceutically relevant or biochemically valuable products.

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