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|Title:||INVESTIGATION OF CONFORMATIONAL DYNAMICS IN ENZYMATIC COMPLEXES BY SINGLE-MOLECULE FLUORESCENCE TECHNIQUE|
|Publisher:||Princeton, NJ : Princeton University|
|Abstract:||Although single-molecule Förster resonance energy transfer (smFRET) has become a powerful technique for dynamic structural biology, there remains challenges in applying smFRET to study the conformational dynamics of complex proteins and their binding complexes. This thesis first describes a series of method developments that expand the toolkit of smFRET. Two categories of methodologies are included: improvement on the capability to capture and characterize biomolecule interactions; and analytical frameworks to robustly quantify the states and the kinetics of state transitions. A multi-function optical microscope that integrates single-molecule and single-particle imaging is first described. This microscope combines multiple illumination modes and detection modes with programmatically controlled switching in between. The new design bridges the timescales covered by current single-molecule fluorescence method. A statistical learning framework capable of determining the states from time series with Gaussian noise is illustrated. By using clustering and Bayesian model selection, this method can extract the number and populations of states, and the kinetics of their transitions without any input kinetic models. Furthermore, an analytical framework is developed to incorporate photobleaching kinetics in the on-/off-time distributions from single-molecule fluorescence experiments. Accounting for the effect of photobleaching allows an accurate quantification of the underlying kinetic parameters. Lastly, a new experimental scheme is demonstrated to facilitate the study of weak interaction between biomolecules using single-molecule approaches. This strategy brings the binding partners in fixed proximity to promote their interactions by co-immobilizing both components on a template molecule. The method is shown to be particularly useful for probing the interactions and conformational dynamics of weak biomolecule complexes. The second part of this thesis focuses on investigating the structural dynamics and protein-protein interactions of nonribosomal peptide synthetases (NRPS), a class of biosynthetic enzymes for antibiotic production in bacteria and fungi. Using single-molecule FRET, kinetic enzymology and mutagenesis, the functional roles of conformations as well as the transitions between them were elucidated. The results provide a mechanistic understanding of the coupling between the conformational changes of the enzyme and the multi-step chemical reactions.|
|Alternate format:||The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu|
|Type of Material:||Academic dissertations (Ph.D.)|
|Appears in Collections:||Chemistry|
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