Sensing conformational dynamics of single trapped proteins in solution
Abstract/Contents
- Abstract
- Proteins are macromolecular nanomachines that perform a wide variety of essential functions in living organisms. Distinct types of proteins in the cell serve as molecular sensors and signalers, sunlight-harvesting antennas and transducers, molecular motors for transport and metabolic energy storage, and oxidizers and reducers of physiological metal ions, to name a few. To achieve these complex functions, proteins are not static building blocks, but instead adopt multiple conformations in distinct functional states. These functions are inherently dynamic, meaning that methods capable of resolving dynamic behavior are necessary to fully elucidate protein function. Many bulk fluorescence methods have been used to study proteins by averaging together the signals from many copies of the same protein. However, such methods fail to capture distributions in protein conformations (e.g. mixtures of active and inactive states) and unsynchronized dynamics among these conformations (e.g. transition rates from inactive to active states under various conditions). Single-molecule fluorescence spectroscopy of proteins, on the other hand, allows direct observation of distributions and dynamics by watching only one protein at a time. In particular, a special device known as the Anti-Brownian ELectrokinetic (ABEL) trap can hold single fluorescently labeled proteins in solution for several seconds of spectroscopic observation without surface attachment, encapsulation, or the use of large beads. The ABEL trap combines fluorescence-based position estimation obtained by scanning a laser spot with the application of electrokinetic feedback forces to counter the Brownian motion of single proteins, one at a time. In this Dissertation I will describe my use of the ABEL trap technique to study dynamics of a variety of biomolecules, with emphasis on two proteins: the [beta]2-adrenergic receptor ([beta]2AR), an essential cellular signaling protein, and the peridinin-chlorophyll-protein (PCP), a sunlight-harvesting pigment-protein complex found in algae. In [beta]2AR, I observed a shift in protein conformation and in time scales of protein dynamics upon binding of an activating drug. In PCP, I observed two distinct classes of conformational change, indicating light-induced conformational flexibility, which may play a physiological role. Ongoing projects include resolving conformational substeps in FoF1 ATP synthase and measuring electron transfer kinetics of the multicopper oxidase Fet3p.
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 | Bockenhauer, Samuel David |
|---|---|
| Associated with | Stanford University, Department of Physics. |
| Primary advisor | Burchat, P. (Patricia) |
| Primary advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Burchat, P. (Patricia) |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Das, Rhiju |
| Thesis advisor | Kobilka, Brian K |
| Advisor | Das, Rhiju |
| Advisor | Kobilka, Brian K |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Samuel David Bockenhauer. |
|---|---|
| Note | Submitted to the Department of Physics. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2013. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2013 by Samuel David Bockenhauer
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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