Fluorescent Biosensors for Characterizing Sugars Will Eventually Be Exported Transporters

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PARK-DISSERTATION-2022.pdf (5.43 MB)
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Park, Jihyun
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School of Chemical and Biomolecular Engineering
School established in 1901 as the School of Chemical Engineering; in 2003, renamed School of Chemical and Biomolecular Engineering
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https://hdl.handle.net/1853/73014
Abstract
Sugars are fundamental to plant physiology: they serve as the main donor of carbon skeleton and act as signaling molecules and osmoprotectants. Sugars Will Eventually be Exported Transporters (SWEETs) are a family of transmembrane transporters key in allocating sugar from source leaves to vital organs throughout the plant. Current technologies to assay SWEETs primarily rely on radiotracer experiments, which originally allowed for sophisticated kinetic experiments in GLUT1, the most well studied of all membrane transporters. However, it took 70 years to reach this deep level of characterization. Genetically encoded activity biosensors have been proposed as an alternative technology for characterization as they can provide locational and dynamic data simultaneously. However, activity biosensors have yet to be readily utilized for plant sugar transporters due to the unclear interpretation of their response and lack of demonstrations showcasing their use for in-depth characterization. The purpose of this thesis is to address these issues through SweetTrac1, a biosensor developed to track Arabidopsis SWEET1 activity. We used the biosensor as a proxy to explore the kinetics and substrate specificity of its wild-type counterpart. By physically defining SweetTrac1’s response and by kinetically modelling the transporter, we found AtSWEET1 to be a symmetric and high-capacity transporter that functions to rapidly equilibrate large concentration gradients of sugar. Through a combination of small molecule screens using SweetTrac1 and cheminformatics, we were also able to discover 12 new substrates and uncover some structural requirements for substrate recognition. The work in this thesis is a demonstration of how activity biosensors can be used to expedite characterization of membrane carriers like SWEETs.
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2022-10-21
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