Engineering and Biological Applications of Turn-on Fluorescent Protein-based Sensors for Chloride
| dc.contributor.advisor | Dodani, Sheel C. | |
| dc.contributor.advisor | Choudhary, Pankaj | |
| dc.contributor.committeeMember | Meloni, Gabriele | |
| dc.contributor.committeeMember | Gassensmith, Jeremiah J. | |
| dc.contributor.committeeMember | Sibert IV, John W. | |
| dc.creator | Tutol, Jasmine Nojadera | |
| dc.date.accessioned | 2023-09-06T18:47:28Z | |
| dc.date.available | 2023-09-06T18:47:28Z | |
| dc.date.created | 2021-08 | |
| dc.date.issued | August 2021 | |
| dc.date.submitted | August 2021 | |
| dc.date.updated | 2023-09-06T18:47:30Z | |
| dc.description.abstract | Chloride is the most abundant anion in our body and is essential for all forms of life. The transport of chloride is linked to cellular functions including cell volume, pH regulation, cell division, muscle contraction, and neuroexcitation. However, dysregulation of cellular chloride transport has been implicated in human diseases such as cystic fibrosis, pancreatitis, and epilepsy suggesting that chloride could be a signal of cellular status. Moreover, we lack a clear molecular-level picture of what chloride is doing. In this thesis, I will discuss the various approaches researchers have used to study chloride in biological systems. Chapter 1 will review the different types of fluorescent proteins that have been used to develop sensors for chloride. To expand on the current state of the art, we have revealed the first examples of standalone turn-on fluorescent protein-based sensors for chloride using the naturally occurring yellow fluorescent protein from the jellyfish Phialidium sp., which is ratiometric and undergoes an excited state proton transfer in the presence of chloride (Chapter 2), and the engineered mNeonGreen protein from the cephalochordate Branchiostoma lanceloatum (Chapter 3). Given that the mNeonGreen sensor operates best at pH 4.5, Chapter 4 will describe how we designed and carried out double site-saturation mutagenesis of noncoordinating residues in the mNeonGreen chloride binding pocket. This protein engineering effort not only improved the chloride sensing properties of mNeonGreen at physiological pH but also generated sensors with the largest turn-on fluorescence responses to chloride thus far (ChlorON). Fluorescence imaging experiments in mammalian cells expressing a ChlorON sensor demonstrate how the advantage of using turn-on fluorescent sensors to provide spatial and temporal resolution for mapping chloride dynamics. Lastly, Chapter 5 will highlight an alternative de novo strategy to convert the membrane-bound, proton-pumping rhodopsin from the cyanobacterium Gloeobacter violaceus (GR) into a non-pumping, red-shifted, and turn-on fluorescent sensor for chloride that can be used to image chloride in bacteria. In this study, we identified how a single point mutation of a key residue in the proton transport pathway can create a new chloride binding site in GR while also altering the protein function and spectroscopic properties to sense chloride. Taken together, this body of work lays the foundation of building protein-based hosts for chloride recognition and illustrates how we can use and adapt naturally occurring proteins to expand the turn-on fluorescent imaging toolkit for chloride that can enable the discovery of new roles for chloride in biology. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | ||
| dc.identifier.uri | https://hdl.handle.net/10735.1/9840 | |
| dc.language.iso | en | |
| dc.subject | Chemistry, Biochemistry | |
| dc.subject | Chemistry, General | |
| dc.title | Engineering and Biological Applications of Turn-on Fluorescent Protein-based Sensors for Chloride | |
| dc.type | Thesis | |
| dc.type.material | text | |
| local.embargo.lift | 08/01/2023 | |
| local.embargo.terms | 08/01/2023 | |
| thesis.degree.college | School of Natural Sciences and Mathematics | |
| thesis.degree.department | Chemistry | |
| thesis.degree.grantor | The University of Texas at Dallas | |
| thesis.degree.name | PHD |
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