Single-Molecule FRET Analyses of NMDA Receptors.
| Autor: | Durham RJ; Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, TX, USA., Jayaraman V; Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center at Houston, Houston, TX, USA. vasanthi.jayaraman@uth.tmc.edu. |
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| Jazyk: | angličtina |
| Zdroj: | Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2024; Vol. 2799, pp. 225-242. |
| DOI: | 10.1007/978-1-0716-3830-9_12 |
| Abstrakt: | Single-molecule fluorescence resonance energy transfer (smFRET) enables the real-time observation of conformational changes in a single protein molecule of interest. These observations are achieved by attaching fluorophores to proteins of interest in a site-specific manner and investigating the FRET between the fluorophores. Here we describe the method wherein the FRET is studied by adhering the protein molecules to a slide using affinity-based interactions and measuring the fluorophores' fluorescence intensity from a single molecule over time. The resulting information can be used to derive distance values for a point-to-point measurement within a protein or to calculate kinetic transition rates between various conformational states of a protein. Comparing these parameters between different conditions such as the presence of protein binding partners, application of ligands, or changes in the primary sequence of the protein can provide insights into protein structural changes as well as kinetics of these changes (if in the millisecond to second timescale) that underlie functional effects. Here we describe the procedure for conducting analyses of NMDA receptor conformational changes using the above methodology and provide a discussion of various considerations that affect the design, execution, and interpretation of similar smFRET studies. (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.) |
| Databáze: | MEDLINE |
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