TY - JOUR
T1 - Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET
AU - Banerjee, Chiranjib
AU - Liauw, Brandon Wey Hung
AU - Vafabakhsh, Reza
N1 - Funding Information:
We thank members of the Reza Vafabakhsh lab for discussions. This work was supported by the National Institutes of Health grant R01GM140272 (to R.V.), by The Searle Leadership Fund for the Life Sciences at Northwestern University, and by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (to R.V.). B.W.L. was supported by the National Institute of General Medical Sciences (NIGMS) Training Grant T32GM-008061.
Publisher Copyright:
© 2022 JoVE Journal of Visualized Experiments.
PY - 2022/8
Y1 - 2022/8
N2 - The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.
AB - The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.
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U2 - 10.3791/64254
DO - 10.3791/64254
M3 - Article
C2 - 36062999
AN - SCOPUS:85137159681
SN - 1940-087X
VL - 2022
JO - Journal of Visualized Experiments
JF - Journal of Visualized Experiments
IS - 186
M1 - e64254
ER -