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|CONFORMATIONAL CONTROL OF REACTIVITY IN HIGHLY FLEXIBLE ENZYMES STUDIED BY SAXS AND CRYO-EM
|Watkins, Maxwell Brady
Protein conformational dynamics
Small-angle X-ray scattering
|Princeton, NJ : Princeton University
|AbstractProteins are molecules fundamentally in motion, and understanding how these motions dictate function at a chemical level is a major challenge in structural biology. Some proteins, such as enzymes involved in the biosynthesis of small molecules, require large domain motions during catalysis and so high levels of flexibility are often intrinsic to these systems. Because of this inherent heterogeneity, these systems are often difficult to study and typically require a multi-faceted approach to begin to describe the relationship between dynamics and function. The work presented in this thesis focuses on the combination of small-angle X-ray scattering (SAXS), single-molecule or single-particle techniques, and computational methods, as a strategy for the study of these highly flexible, heterogeneous enzyme systems. A main focus of this thesis is the cobalamin-dependent methionine synthase (MetH), which performs a cobalamin-mediated, multi-step reaction to produce the amino acid methionine. MetH is one of only two cobalamin-utilizing enzymes in humans, and although its biochemistry has been studied extensively the domain rearrangements required for its chemical processes have been challenging to visualize. Using SAXS and cryo-electron microscopy (cryo-EM), we present the first depiction of the enzyme in its entirety. We further propose a model for how substrates govern the conformational dynamics involved in the MetH catalytic and reactivation cycles. Additional work presented in this thesis demonstrates the combined use of SAXS, single-molecule Förster resonance energy transfer (FRET), and computation to study the conformational cascade of a similarly flexible non-ribosomal peptide synthetase (NRPS) module, Gramicidin synthase A (GrsA). NRPSs are complex, dynamic enzymes often involved in the biosynthesis of small-molecule secondary metabolites with medical relevance. Overall, the work presented in this thesis yields new insights into the large-scale domain motions required for the chemical function of both MetH and GrsA and also provides a general approach for the study of highly flexible enzyme systems.
|Type of Material:
|Academic dissertations (Ph.D.)
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