FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
| Autor: | Daniel S. Terry, Scott C. Blanchard, Changhao He, Xiaoyu Wang, Gabriel A. Fitzgerald, Olga Boudker, Didar Ciftci, Gerard H. M. Huysmans, Zhou Zhou |
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| Rok vydání: | 2021 |
| Předmět: |
chemistry.chemical_classification
education.field_of_study Total internal reflection fluorescence microscope Chemistry Strategy and Management Mechanical Engineering Population Metals and Alloys Periplasmic space Industrial and Manufacturing Engineering Amino acid Förster resonance energy transfer Membrane Symporter Methods Article Biophysics Energy source education |
| Zdroj: | Bio Protoc |
| ISSN: | 2331-8325 |
| Popis: | Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter Glt(Ph) as a model system. We prepare proteo-liposomes containing reconstituted purified Glt(Ph) transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population. |
| Databáze: | OpenAIRE |
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