Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

Summary

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

Abstract

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.

Introduction

The transfer of information across the plasma membrane is heavily dependent on the function of membrane receptors¹. Ligand binding to a receptor leads to a conformational change and receptor activation. This process is often allosteric in nature². With over 800 members, G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans³. Due to their role in nearly all cellular processes, GPCRs have become important targets for therapeutic development. In the canonical model of GPCR signaling, agonist activation results in conformational changes of the receptor that subsequ....

Protocol

The overall workflow of the protocol is described in Figure 1.

1. Preparation of the sample chamber

1. Slide and coverslip cleaning
   NOTE: These steps aim to clean the surfaces of the slides as well as the coverslips and prepare them for aminosilanization. One critical requirement for conducting single-molecule fluorescence experiments on surface-tethered molecules is a passivated surface. The most reliable and reproducible passivation technique involves covalently attaching inert polymer chains to the glass surface as a dense layer. Polyethylene glycol (PEG) is the most efficient polymer used ....

Results

Expression and fluorescent labeling of UAA-based FRET sensor
Herein, exemplary results of the insertion and fluorescent labeling of a UAA (AZP) within the CRD of mGluR2 (548UAA) are discussed¹¹. As mentioned previously, to insert AZP into mGluR2, co-expression of the engineered translational machinery, which includes a modified tRNA synthetase and complementary tRNA (pIRE4-Azi), and mGluR2 containing an amber codon at position 548, created using mutagenesis, is necessary (

Discussion

GPCRs are proteins that operate on the cell membrane to initiate signal transduction. Many GPCRs consist of multiple domains, with signaling being dependent on the cooperative interaction between the domains. To modulate the properties of these membrane receptors, it is essential to understand the dynamic behavior of the multiple domains. Single-molecule fluorescence resonance energy transfer (smFRET) is a fluorescence technique that enables the measurement of protein conformation and dynamics in real time

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(+)-Sodium L-Ascorbate	Sigma Aldrich	Cat # 11140-250G
4-azido-L-phenylalanine	Chem-Impex International	Cat # 06162
548UAA	Liauw et al. 2021		Transfected construct
Acetic Acid	Fisher Chemical	64-19-7
Acetone	Fisher Chemical	67-64-1
Adobe Illustrator (2022)	https://www.adobe.com/	RRID:SCR_010279	Software, algorithm
Aminoguanidine (hydrochloride)	Cayman Chemical	81530
Aminosilane	Aldrich	919-30-2
Bath Sonicator 2.8 L	Fisher Scientific		Ultrasonic Bath 2.8 L
Biotin-PEG	Laysan Bio Inc	Item# Biotin-PEG-SVA-5000-100mg
BTTES	Click Chemistry Tools	1237-500
Copper (II) sulfate	Sigma Aldrich	Cat # 451657-10G
Cover slip	VWR	16004-306	Sample chamber
Cy3 Alkyne	Click Chemistry Tools	TA117-5
Cy5 Alkyne	Click Chemistry Tools	TA116-5
DDM	Anatrace	Part# D310 1 GM	Detergent
DDM-CHS (10:1)	Anatrace	Part# D310-CH210 1 ML	Detergent with cholecterol
Defined Fetal Bovine Serum	Thermo Fisher Scientific	SH30070.03
Di01-R405/488/561/635	Semrock		Notch filter
DMEM	Corning	10-013-CV
EMCCD	Andor	DU-897U	Camera
ET542lp	Chroma		Long pass emission filter
FF640-FDi01	Semrock		Emission dichroic filter
FLAG-tag antibody	Genscript	A01429
Fluorescent bead	Invitrogen T7279	TetraSpeck microspheres	Spherical bead
Glass slides	Fisherfinest	12-544-4	sample chamber
Glutamate	Sigma Aldrich	Cat # 6106-04-3
HEK 293T	Sigma Aldrich	Cat # 12022001	Cell line
HEPES	FisherBioReagents	7365-45-9
Image splitter	OptoSplit II
KOH	Fluka	1310-58-3
Laser	Oxxius		4-line laser combiner
Lipofectamine 3000 Transfection Reagent	Thermo Fisher Scientific	L3000015	Transfection Reagent
Methanol	Fisher Chemical	67-56-1
Microscope	Olympus	Olympus IX83
Milli-Q water	Barnstead		Water Deionizer
m-PEG	Laysan Bio Inc	Item# MPEG-SIL-5000-1g
NF03-405/488/532/635	Semrock		Dichroic mirror
OptiMEM	Thermo Fisher Scientific	51985091	Reduced Serum Medium
OptiMEM/Reduced serum medium	Thermo Fisher Scientific
OriginPro (2020b)	https://www.originlab.com/	RRID:SCR_014212	Data analysis and graphing software
Penicillin-Streptomycin	Gibco	15140-122
pIRE4-Azi	Addgene	Plasmid # 105829	Transfected construct
Poly-L-lysine hydrobromide	Sigma Aldrich	Cat # P2636
Protocatechuic acid (PCA)	HWI group	99-50-3
smCamera (Version 1.0)	http://ha.med.jhmi.edu/resources/		Camera software
Sodium bicarbonate	FisherBioReagents	144-55-8
Sodium hydroxide (NaOH)	Sigma	1310-73-2
Syringe filter	Whatman UNIFLO	Cat#9914-2502	Liquid filtration
Trolox	Sigma	53188-07