Dynamics of DNA gyrase : species differences and antibiotic inhibition mechanisms
Abstract/Contents
- Abstract
- Single-molecule techniques have been used to uncover the biophysical mechanisms of antimicrobial targets and their mechanisms of inhibition, and could make important contributions to future drug development pipelines. DNA gyrase is an essential nucleoprotein motor in all bacteria and a major target for the treatment of Mycobacterium tuberculosis (TB) and many other pathogens. Gyrase hydrolyzes ATP to add negative supercoils to DNA, employing a strand passage mechanism that has been previously investigated using biophysical and biochemical approaches, including single-molecule rotor bead tracking (RBT). We have now used RBT to measure rates, processivities, and conformational distributions and dynamics of gyrase from three evolutionarily distant, pathogenically relevant bacterial species: Escherichia coli (EC), TB, and Clostridium difficile (CD). Supercoiling is seen to be processive in all cases, with TB gyrase displaying velocities ~5X slower than EC. Compared with EC, TB gyrase more strongly populates a critical intermediate in which DNA is wrapped around the enzyme and poised for directional strand passage. Our substep measurements reveal some universal states, along with differences in populations and transition rates that may reflect distinct cellular requirements for biophysical properties across species. In preliminary work, we also demonstrate the use of RBT to observe inhibited states of gyrase with example drugs from major classes of antibiotic inhibitors, including widely prescribed fluoroquinolones and recently developed novel bacterial topoisomerase inhibitors (NBTIs). This work shows that RBT can be used to study the dynamics of drug inhibition of DNA gyrase. Further experiments complemented by high-resolution structural characterization of conformational states may help develop improved and novel inhibitors. This dissertation also describes an effort to develop curiosity-driven interdisciplinary science capabilities outside the university setting to promote access and inclusion to science, technology, engineering, and mathematics professions (STEM). We present an outreach program called Future Advancers of Science and Technology (FAST), which has partnered over 200 high school students with over 100 Stanford graduate students to work on year-long science and engineering projects that investigate unknown questions or invent new devices. Previous programs have studied the positive effects of project-based learning and long-term mentorship separately. In FAST, we combined these two approaches to STEM education. Case studies of students who chose to complete biophysics-related projects illustrate anecdotally how student-driven projects can be developed in a relatively resource-limited setting, with positive impacts on students' career growth. We have developed a plan for more rigorously assessing the impact of the program on mentees and mentors in the next cycle.
Description
| 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 | 2019; ©2019 |
| Publication date | 2019; 2019 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Galvin, Cooper J |
|---|---|
| Degree supervisor | Bryant, Zev David |
| Thesis advisor | Bryant, Zev David |
| Thesis advisor | Huang, Kerwyn Casey, 1979- |
| Thesis advisor | Kim, Peter, 1958- |
| Degree committee member | Huang, Kerwyn Casey, 1979- |
| Degree committee member | Kim, Peter, 1958- |
| Associated with | Stanford University, Biophysics Program. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Cooper Galvin. |
|---|---|
| Note | Submitted to the Biophysics Program. |
| Thesis | Thesis Ph.D. Stanford University 2019. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Cooper J Galvin
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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