High-speed multimodal microscopy for studying structural dynamics of biomolecular machines
Abstract/Contents
- Abstract
- Single molecule tracking and manipulation is now an established approach for interrogating the dynamics of biomolecular systems, both in vivo and in vitro. By reporting nanoscale displacements of functioning complexes, constraints can be placed on structural rearrangements (such as in motor activity) as well as transition kinetics. This information is obtained directly from individual molecules, permitting branched pathways and statistical distributions to be observed rather than bulk means. However, single molecule experiments can exhibit low dimensionality and poor signal-to-noise. In particular, the spatiotemporal resolution of DNA twist measurements has been limited by the use of angular probes with high rotational drag, which prevents detection of short-lived intermediates or small angular steps. We introduce gold rotor bead tracking (AuRBT), which yields > 100× improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, which are monitored by instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. Our analysis of high-speed structural dynamics of the enzyme DNA gyrase using AuRBT revealed an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with > 50× shorter integration times than previous techniques; we demonstrated high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence. Additionally, we have augmented our system with synchronous single fluorophore imaging, in order to detect the binding of DNA, proteins, or ligands to mechanochemical substates identified by highspeed tracking. We have validated the use of FRET in combination with rotor bead tracking as a tool to probe intramolecular dynamics simultaneously with mechanical degrees of freedom, generating multidimensional data that reports on the highspeed structural dynamics of functioning molecular machines.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2014 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lebel, Paul Martin |
|---|---|
| Associated with | Stanford University, Department of Applied Physics. |
| Primary advisor | Bryant, Zev David |
| Primary advisor | Mabuchi, Hideo |
| Thesis advisor | Bryant, Zev David |
| Thesis advisor | Mabuchi, Hideo |
| Thesis advisor | Greenleaf, William James |
| Advisor | Greenleaf, William James |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Paul Martin Lebel. |
|---|---|
| Note | Submitted to the Department of Applied Physics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2014. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Paul Martin Lebel
- License
- This work is licensed under a Creative Commons Attribution Non Commercial No Derivatives 3.0 Unported license (CC BY-NC-ND).
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