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dc.creatorFraser, Alec
dc.date.accessioned2021-12-02T16:44:25Z
dc.date.available2021-12-02T16:44:25Z
dc.date.created2021-12
dc.date.submittedDecember 2021
dc.identifier.urihttps://hdl.handle.net/2152.3/11356
dc.description.abstractOur understanding of the mechanism of protein function has been continuously adapted to be consistent with new experimental and theoretical findings. Eventually, the idea of a relationship between the structure and function of proteins emerged. As the methods available to probe structure became higher-throughput, more precise and easier to use, the conceptual underpinnings of this relationship have grown broader and more wholistic. Our comprehension of protein structure has shifted from static globular structures towards dynamic ensembles of folded and unfolded states. Despite this paradigm shift, existing methods for the characterization of large macromolecules remain focused on the characterization of stable, low-energy states. In this work, we aim to characterize the structure, and function, of various macromolecular complexes beyond the conventional low-energy, singular state approach. To do this, we extend existing methods and create new approaches catered for the study of large-scale macromolecular dynamics. Here, we characterize the disorder-to-order transition of a 5-subunit bacteriophage AR9 RNA polymerase holoenzyme throughout the process of uracil-specific promoter recognition with cryo-EM and x-ray crystallography. Using this structural information, we assess the energetics of DNA binding to the promoter binding pocket via molecular dynamics. Furthermore, energetic, and structural insights enable the design of a mutant RNAP holoenzyme which can recognize altered thymine-containing DNA templates. Following this, we describe the mechanism of pyocin contraction using a combination of purpose-made computational modelling, electron microscopy and biophysical assays. Together, we characterize the relevant energetics and forces generated throughout the contraction process. Moreover, we validate predictions on how the energetics of contraction can be manipulated through mutagenesis. Finally, using cryo-EM, we solve the structures of the bacteriophage A511 sheath-tube complex in the extended, contracted, and intermediate states. Using this novel structural information, we assess the relationship between the motions of sheath subunits during contraction. We find that the process of sheath contraction can be described by a combination of local, subunit-level linear and non-local global properties. With the low-energy, tertiary structure protein folding problem largely solved, we propose that similar dynamical methods to those described below can be used to further our wholistic understanding of the structure function relationship.
dc.format.mimetypeapplication/pdf
dc.subjectStructural Biology, CryoEM
dc.titleIntegration of Structural Methods to Characterize the Dynamics of Macromolecular Complexes
dc.typeThesis
dc.date.updated2021-12-02T16:44:27Z
dc.type.materialtext
thesis.degree.nameMolecular Biophysics Educational Track (Doctoral)
thesis.degree.levelDoctoral
thesis.degree.grantorThe University of Texas Medical Branch at Galveston
thesis.degree.departmentBiochemistry and Molecular Biology


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