Using semi-synthetic fluorescent proteins to understand proton transfer
Abstract/Contents
- Abstract
- Proton transfer is a fundamental process in biology, essential to critical protein functions including energy transduction and enzymatic catalysis. Green fluorescent protein (GFP) has proven to be a powerful tool in cellular biology by virtue of its autocatalytically formed fluorescent chromophore. In this thesis I utilize the chromophore's optical sensitivity to protonation state, its unique chemical properties, and GFP's robust and modular structure for a detailed look into proton transfer and the role played by the surrounding protein environment. Towards this goal we have employed semi-synthetic methodology including the introduction of synthetic peptides, isotope labels, and unnatural amino acids in order to probe and perturb the underlying chemical processes. In Chapter 2 we develop a new method using a combination of circular permutation, site-specific proteolysis, and synthetic peptide substitution to efficiently replace any structural element of GFP. This includes the first-ever catalysis of chromophore maturation in an artificial substrate. I further use this method to examine large electrostatic perturbations not previously accessible to the chromophore environment through the use of a novel helix transplantation strategy. Chapter 3 introduces a site-specific 13C isotope label into the chromophore with a large chemical shift dependence on the protonation state. The resulting 1D 13C-NMR spectra reflected the dynamic characteristics of the chemical exchange but stood in apparent contradiction to timescales inferred from fluorescence correlation spectroscopy (FCS). The discrepancies were resolved by a two-site model of proton transfer which clarifies the factors governing the extent of internal coupling between ionization sites and identifies [beta]-strand 7 as playing a critical structural role in gating proton transfer with the solution. Chapter 4 addresses the controversial topic of the role (or even existence) of low-barrier hydrogen bonds (LBHBs) in proteins by closely examining a putative LBHB in the GFP mutant S65T, H148D. Unnatural halide-substituted tyrosines were incorporated into the chromophore and the attendant changes in pKa matching with Asp148 were studied with structural, spectroscopic, and equilibrium isotope methods. Analysis of the data with a 1D model of hydrogen bonding lead us to conclude that the interaction is not an LBHB despite meeting the requisite criteria of a short O-O distance and near-perfect pKa matching between donor and acceptor sites. The implication of these results is that LBHBs may be generally difficult to attain in proteins and thus are likely not responsible for a significant degree of transition state stabilization in enzymes relative to short ionic hydrogen bonds. In summary, we have measured proton transfer processes on a wide range of timescales from gross structural motion occurring over tens of milliseconds down to possible proton delocalization across a short hydrogen bond. This work demonstrates the versatility of GFP as model system for exploring these reactions especially when combined with modern semi-synthetic technologies. The insights thus obtained may be applied toward refining the general understanding of proton transfer in proteins.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Oltrogge, Luke McDonald |
|---|---|
| Associated with | Stanford University, Department of Chemistry. |
| Primary advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Boxer, Steven G. (Steven George), 1947- |
| Thesis advisor | Martinez, Todd J. (Todd Joseph), 1968- |
| Thesis advisor | Rao, Jianghong |
| Advisor | Martinez, Todd J. (Todd Joseph), 1968- |
| Advisor | Rao, Jianghong |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Luke McDonald Oltrogge. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2015 by Luke McDonald Oltrogge
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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