Mapping microbial transporters with small molecule fluorescent probes
EMSL Project ID
60840
Abstract
Objectives: There is a gap in our ability to measure transport of cell materials (sugars, lipids, amino acids, metabolites, cofactors, etc.) across membranes, and to observe transporter localization dynamics in microbes. Similarly, transporter quantification can be misrepresented in global proteomics, and membrane-associated proteomics may still lack functional annotation. We need a comprehensive set of methods for profiling transporter identity and function in microbial systems. In this project, we propose to profile transporter proteins using a substrate mimicking affinity-probing approach. The objectives for this proposal are to demonstrate the modular utility of activity-based probes (ABPs) to: (i) visualize transporter activity and localization in live cells, and (ii) identify transporter function. Specifically, a dual function fluorescent mannose-directed ABP—one function that shows free mannose movement and another that can be linked to the transporter pore—will be used to quantify substrate uptake (measured by intracellular signal increase) and visualize transporter localization at the membrane surface in live yeast. A successful outcome will serve as a foundation for expanding ABP capabilities toward functional identification of various organic carbon and nitrogen (lipids, peptides, sugars, etc.) transport proteins. This set of experiments will serve as a basis for engaging users to develop probes specific to users’ need that can be developed through other EMSL user grants.
Project Details
Start Date
2023-04-17
End Date
2024-01-14
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)