Structural insight into the photochemistry of split green fluorescent proteins
Abstract/Contents
- Abstract
- Previous studies in the Boxer lab characterized two variants of split green fluorescent protein (GFP), which appeared to behave distinctly with respect to interaction with their complementary peptides in the presence of light. Truncated green fluorescent protein (GFP) with its 11th β-strand removed (i.e. the strand 11 system) can recover the absorbance and fluorescence properties of the whole protein when refolded in the presence of synthetic strand 11 peptide. When refolded on its own, the same truncated protein is no longer receptive to binding exogenous strand 11; however, light irradiation generates a species which is capable of binding the complementary peptide. Meanwhile, the truncated GFP with its 10th β-strand removed (i.e. the strand 10 system) can readily bind to synthetic strand 10 peptide to recover the absorbance and the fluorescence of the whole protein. It was found that strand 10 spontaneously dissociates from this bound complex very slowly, and this process is greatly accelerated by light irradiation. In short, the strand 11 system undergoes photoassociation, and the strand 10 system undergoes photodissociation, but the origin of this difference was unclear despite extensive characterization of the two species. Our work attempted to reconcile the apparent differences in the photophysical properties of the two systems by directly investigating the structure of the strand 11 system at atomic level resolution using two techniques: solution state NMR and X-ray crystallography. Although we fell short of obtaining a full NMR structure, we were able to assign backbone chemical shifts for most of the residues in two variants of the strand 11 system, and probed the solvent accessibility of different residues by hydrogen-deuterium exchange. In the process of preparing this protein for crystallography, we found that the attached N-terminal His-tag, which is generally assumed to have negligible impact on the properties of the fused protein, was necessary to preserve the stability and spectral properties of the truncated protein. In the crystal structure of this construct, we found that the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides serves to reconcile many of the apparent differences between the peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2017 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Deng, Alan |
|---|---|
| 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 | Lin, Michael Z |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- |
| Advisor | Lin, Michael Z |
| Advisor | Moerner, W. E. (William Esco), 1953- |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Alan Deng. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2017. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2017 by Alan Deng
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...