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. 2021:652:193-212.
doi: 10.1016/bs.mie.2021.02.005. Epub 2021 Mar 9.

Single molecule FRET methodology for investigating glutamate receptors

Nabina Paudyal  1 , Nidhi Kaur Bhatia  2 , Vasanthi Jayaraman  3
Affiliations

Single molecule FRET methodology for investigating glutamate receptors

Nabina Paudyal et al. Methods Enzymol. 2021.

Abstract

Single molecule Förster Resonance Energy Transfer (smFRET) allows us to measure variation in distances between donor and acceptor fluorophores attached to a protein, providing the conformational landscape of the protein with respect to this specific distance. smFRET can be performed on freely diffusing molecules or on tethered molecules. Here, we describe the tethered method used to study ionotropic glutamate receptors, which allows us to track the changes in FRET as a function of time, thus providing information on the conformations sampled and kinetics of conformational changes in the millisecond to second time scale. Strategies for attaching fluorophores to the proteins, methods for acquiring and analyzing the smFRET trajectories, and limitations are discussed.

Keywords: Conformational landscape; Efficiency histograms; Fluorophores; Ionotropic glutamate receptors; smFRET.

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Figures

Figure 1.
Figure 1.
Panel A. Spectral profile of donor (Alexa 555) and acceptor (Alexa 647). Spectral overlap region is shown in yellow and lies between donor emission (solid magenta) and acceptor excitation (dotted blue). Panel B. FRET efficiency as a function of the distance between donor and acceptor fluorophores (R). In the given equation, R0 represents the Forster’s distance at which the FRET efficiency (E) is 50%. Forster’s distance value for Alexa 555 and Alexa 647 is 51Å.
Figure 2.
Figure 2.
Side view of an iGluR tetramer showing subunit and domain arrangement in left panel. The right panel shows the top-down view of amino-terminal domain, agonist-binding domain and transmembrane domain depicting the sites chosen for labeling for smFRET experiments. In a multidomain protein like iGluR, one domain is labeled at a time at sites highlighted in magenta to study the conformational changes induced in that particular region of a full-length receptor. Structure image was created using Visual Molecular Dynamics (VMD) software.
Figure 3.
Figure 3.
Schematic outline of the smFRET protocol involving A) sample preparation, B) slide preparation, C) data acquisition, and D) data analysis.
See this image and copyright information in PMC

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