Dissecting structure-function relationships in molecular motors using protein engineering and single-molecule methods

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Biological cells can harness the free energy of ATP hydrolysis to perform mechanical tasks using molecular motor proteins. These nanoscale machines are able to generate directional motion through mechanochemical cycles which rely on allosteric communication and large rearrangements of protein domains. In studies of molecular motors, protein engineering allows us to test our understanding of relationships between structure and function, while single-molecule methods allow us to directly observe motor dynamics. Here we consider two systems which undergo large conformational changes: cytoplasmic dynein and DNA gyrase. We use protein engineering to investigate structural features that contribute to dynein velocity and processivity. Building on our initial findings, we are able to design dynein motors that change speed in response to light. The speed and controllability of future designs may be improved with further engineering, in order to generate light-activatable, dynein-based tools which can be used to study transport functions in vivo. In the second half of this dissertation, we consider a single-molecule technique for multimodal measurements of mechanics and fluorescence in DNA and DNA:protein complexes. Mechanical measurements based on magnetic tweezers are combined with simultaneous fluorescence imaging that can report on macromolecular binding and local conformational changes. We outline how this method can be applied to study the mechanism of DNA gyrase, a motor which introduces negative supercoils by coordinating protein domain motions and ATP hydrolysis with DNA cleavage and religation. We observe binding coincident with mechanics and report on challenges in using FRET-labeled enzymes to correlate domain motions with mechanical substeps. We anticipate that correlative multimodal measurements will be valuable tools for characterizing the dynamics of DNA gyrase and other large nucleoprotein machines.


Type of resource text
Form electronic resource; remote; computer; online resource
Extent 1 online resource.
Place California
Place [Stanford, California]
Publisher [Stanford University]
Copyright date 2022; ©2022
Publication date 2022; 2022
Issuance monographic
Language English


Author Ierokomos, Athena
Degree supervisor Bryant, Zev David
Thesis advisor Bryant, Zev David
Thesis advisor Fordyce, Polly
Thesis advisor Huang, Possu
Degree committee member Fordyce, Polly
Degree committee member Huang, Possu
Associated with Stanford University, Biophysics Program


Genre Theses
Genre Text

Bibliographic information

Statement of responsibility Athena Ierokomos.
Note Submitted to the Biophysics Program.
Thesis Thesis Ph.D. Stanford University 2022.
Location https://purl.stanford.edu/tz186kh3516

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© 2022 by Athena Ierokomos

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