Observing protein machines in action : foundations for a chemical approach
Abstract/Contents
- Abstract
- The conformational gymnastics and specific interactions of individual proteins drive the majority of intracellular processes. Yet detailed, dynamic information for these interactions and conformational changes is difficult to obtain and in most cases remains unavailable or inaccessible. In this work, we develop and validate methodology to rapidly obtain global coverage of residue specific information about a protein's conformation, under a set of conditions or after initiation of an event. Cysteine mutations are used as probes of solvent accessibility and changes in modification rate serve as a corollary for changing conformation. To allow rapid generation of this cysteine modification platform, we first increased the robustness and scope of our cysteine misincorporation technology to the entire E. coli genetic code. This allowed for controlled, low frequency cysteine insertion at every position. Second, to demonstrate the power of cysteine modification to determine lightly populated, equilibrium conformations, we used thiol exchange to identify the high energy unfolding intermediates of RNase H D10A. Finally, to elucidate conformational changes that occur on the millisecond time scale, we developed technology and validated chemistry to take 2 millisecond snapshots of a protein's solvent accessibility through rapid pulse/quench cysteine modification.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2012 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Schmidt, Kierstin Liese |
|---|---|
| Associated with | Stanford University, Department of Chemistry |
| Primary advisor | Harbury, Pehr |
| Thesis advisor | Harbury, Pehr |
| Thesis advisor | Herschlag, Daniel |
| Thesis advisor | Khosla, Chaitan, 1964- |
| Thesis advisor | Straight, Aaron, 1966- |
| Advisor | Herschlag, Daniel |
| Advisor | Khosla, Chaitan, 1964- |
| Advisor | Straight, Aaron, 1966- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Kierstin Liese Schmidt. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Ph.D. Stanford University 2012 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2012 by Kierstin Liese Schmidt
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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