Single-Molecule FRET Analyses of NMDA Receptors

Authors

Ryan J Durham
Vasanthi Jayaraman

Language

English

Publication Date

1-1-2024

Journal

Methods in Molecular Biology

DOI

10.1007/978-1-0716-3830-9_12

PMID

38727910

PMCID

PMC11164542

PubMedCentral® Posted Date

1-1-2025

PubMedCentral® Full Text Version

Author MSS

Abstract

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.

Keywords

Fluorescence Resonance Energy Transfer, Receptors, N-Methyl-D-Aspartate, Single Molecule Imaging, Protein Conformation, Kinetics, Fluorescent Dyes, Humans, Protein Binding, Single-Molecule Methods, Fluorescence Resonance Energy Transfer, Protein Dynamics, NMDA Receptors, Glutamate Receptors

Published Open-Access

yes

Recommended Citation

Ryan J Durham and Vasanthi Jayaraman, "Single-Molecule FRET Analyses of NMDA Receptors" (2024). Faculty, Staff and Student Publications. 5906.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/5906