Molecular engineering of monomeric red fluorescent proteins for biosensing applications
Abstract/Contents
- Abstract
- Fluorescent proteins (FPs) have become indispensable tools that revolutionized the study of biology. High resolution crystal structures have allowed us to understand how structure and residue positioning within the tight FP barrel relates to a specific photo or biophysical function. These properties allow us to engineer FPs with unique spectral and photochemical properties such as for brightness, maturation, photostability, and monomericity. Further manipulation of FPs based on their structure and function can enable engineering them into novel biosensors. In my thesis work, I present two new beta-barrel FPs and demonstrate their use in biosensing applications. I will also describe engineering a new class of FPs from scratch based on an enzyme. I first show the engineering and development of a monomeric cyan excitable RFP (mCyRFP1) from its dimeric predecessor CyOFP1. mCyRFP1's unique properties, including monomericity, high brightness, large Stokes shift, and large emission separation from EGFP, enables for single excitation dual wavelength imaging with green fluorescent indicators such as GCaMP6 in brain slices and in vivo. Additionally, we show that mCyRFP1 can be used as an efficient FRET donor to a far-red FP mMaroon1 due to its high quantum yield and high maturation efficiency. We show that mCyRFP1-mMaroon1 can be used for dual-color fluorescence lifetime imaging (FLIM)-FRET to image single dendritic spines undergoing structural plasticity. Secondly, I describe engineering a second generation mCyRFP1 into mCyRFP2. mCyRFP2 displays enhanced photophysical properties, including improved brightness and photostability, making it almost 2-fold brighter than mCyRFP1. mCyRFP2's improved and more complete maturation enables it to be used as an efficient fusion to a green voltage indicator, ASAP3 as a reference protein to correct for motion artifacts. We show that ASAP3-mCyRFP2 is unique in retaining efficient membrane targeting while allowing single wavelength excitation dual emission separation of both fluorophores in neurons. We further demonstrate that ASAP3-mCyRFP2 can be better used to report voltage changes using the green/red ratio than ASAP3 alone in beating cardiomyocytes. Lastly, I describe the first attempts to engineer a new class of near-infrared (NIR) fluorescent proteins based on the enzyme phycocyanobilin-ferredoxin oxidoreductase enzyme (PcyA) that oxidizes biliverdin to phycocyanobilin. I demonstrate that we can create an originally non-fluorescent enzyme into an NIR FP abolishing the enzymatic activity of PcyA while preserving PcyA's ability to bind to BV
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2020; ©2020 |
| Publication date | 2020; 2020 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Kim, Bongjae Benjamin |
|---|---|
| Degree supervisor | Lin, Michael Z |
| Thesis advisor | Lin, Michael Z |
| Thesis advisor | Bryant, Zev David |
| Thesis advisor | Huang, Possu |
| Degree committee member | Bryant, Zev David |
| Degree committee member | Huang, Possu |
| Associated with | Stanford University, Department of Bioengineering. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Benjamin B. Kim |
|---|---|
| Note | Submitted to the Department of Bioengineering |
| Thesis | Thesis Ph.D. Stanford University 2020 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2020 by Bongjae Benjamin Kim
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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