Advanced single molecule fluorescence analysis and force spectroscopy to understand myosin motors
Abstract/Contents
- Abstract
- Myosins are actin-based molecular motor proteins that are associated with numerous cellular functions both in muscle and non-muscle cells. In the past decades, single molecule biophysics has provided many useful in-vitro techniques that enable one to understand detailed molecular mechanisms of myosin-actin interaction. I will describe our results and continuing efforts in (i) single-molecule fluorescence localization analysis with TIRF microscopy, and (ii) single-molecule force spectroscopy with optical traps to understand myosin motors. Our theoretical point-spread function combined with maximum-likelihood-estimation precisely extracts both positions and orientations of fixed fluorescent molecules simultaneously. This approach is now mature as a structural tool, and I have applied it to two examples: myosin V labeled with a fixed dye walking on actin filaments, and dsDNA with two differently colored probes doubly attached to its backbone. We experimentally show that our method is not only precise but also accurate. Familial hypertrophic cardiomyopathies (HCM) are common genetic heart diseases that are often caused by single point mutations, especially in human beta cardiac myosin II. Using an optical trap assay, we found that the human beta cardiac myosin with the R453C HCM-causing mutation produces significantly elevated intrinsic force. We further developed a new method called harmonic force spectroscopy that can extract a force-velocity curve from a single cardiac myosin molecule. We found that a strong-to-weak transition of a cardiac myosin bound to an actin filament is modulated by an external load, which can be explained by simple Arrhenius transition theory.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2013 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Sung, Jongmin |
|---|---|
| Associated with | Stanford University, Department of Applied Physics. |
| Primary advisor | Moerner, W. E. (William Esco), 1953- |
| Primary advisor | Spudich, James A |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Spudich, James A |
| Thesis advisor | Das, Rhiju |
| Advisor | Das, Rhiju |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Jongmin Sung. |
|---|---|
| Note | Submitted to the Department of Applied Physics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2013. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2013 by Jong Min Sung
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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