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.

<ol>
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The authors declare no competing interests.

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<sup class="xre...

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 subsequently activate the heterotrimeric G protein complex via exchange of GDP for GTP at the nucleotide binding pocket of G&#945;. The activated G&#945;-GTP and G&#946;&#947; subunits then control the activity of downstream effector proteins and propagate the signaling cascade4,5. This signaling process essentially depends on the ability of ligands to change the three-dimensional shape of the receptor. A mechanistic understanding of how ligands achieve this is critical for developing new therapeutics and designing synthetic receptors and sensors.
Metabotropic glutamate receptors (mGluRs) are members of the class C GPCR family and are important for the slow neuromodulatory effects of glutamate and tuning neuronal excitability6,7. Among all GPCRs, class C GPCRs are structurally unique in that they function as obligate dimers. mGluRs contain three structural domains: the Venus flytrap (VFT) domain, cysteine-rich domain (CRD), and transmembrane domain (TMD)8. The conformational changes during the activation process are complex and involve local and global conformational coupling that propagate over a 12 nm distance, as well as dimer cooperativity. The intermediate conformations, temporal ordering of states, and rate of transition between states are unknown. By following the conformation of individual receptors in real time, it is possible to identify the transient intermediate states and the sequence of conformational changes during activation. This can be achieved by applying single-molecule fluorescence resonance energy transfer9,10 (smFRET), as was recently applied to visualize the propagation of conformational changes during the activation of mGluR211. A key step in FRET experiments is the generation of FRET sensors by site-specific insertion of the donor and acceptor fluorophores into the protein of interest. An unnatural amino acid (UAA) incorporation strategy was adopted12,13,14,15 to overcome the limitations of typical site-specific fluorescent labeling technologies that require the creation of cysteine-less mutants or the insertion of a large genetically encoded tag. This allowed the conformational rearrangement of the essential compact allosteric linker, which joined the ligand-binding and signaling domains of mGluR2, to be observed. In this protocol, a step-by-step guide to performing smFRET experiments on mGluR2 is presented, including the approach for site-specific labeling of mGluR2 with UAA to attach fluorophores using the copper-catalyzed azide cyclization reaction. Moreover, this protocol describes the methodology for the direct capture of membrane proteins and data analysis. The protocol outlined here is also applicable to studying the conformational dynamics of other membrane proteins.

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

visualizing the conformational dynamics of membrane receptors using single-molecule fret

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.

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 (<s...

Watch this Scientific Journal Video about Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET at JoVE.com

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

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.

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

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3.1K Views.  Northwestern University. 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.

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

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a determinant phosphoinositide of the plasma membrane (PM), and it is required to modulate ion channels, actin dynamics, exocytosis, endocytosis, intracellular signaling, and many other cellular processes. However, the spatial organization of PI(4,5)P2 in the PM is controversial, and its nanoscale distribution is poorly understood due to the technical limitations of research approaches. Here by utilizing single molecule localization microscopy and the Pleckstrin Homology (PH) domain based dual color fluorescent probes, we describe a novel method to visualize the nanoscale distribution of PI(4,5)P2 in the PM in fixed membrane sheets as well as live cells.

PI(4,5)P2 regulates various cellular functions, but its nanoscale organization in the cell plasma membrane is poorly understood. By labeling PI(4,5)P2 with a dual-color fluorescent probe fused to the Pleckstrin Homology domain, we describe a novel approach to study the PI(4,5)P2 spatial distribution in the plasma membrane at the nanometer scale.

Phosphoinositides in the cell membrane are signaling lipids with multiple cellular functions. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a ...

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the &#181;s-ms Timescale

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important biological processes. Understanding how the balance between structure and dynamics governs biological function is a new frontier in modern structural biology and has ignited several technical and methodological developments. Among these, CPMG relaxation dispersion solution NMR methods provide unique, atomic-resolution information on the structure, kinetics, and thermodynamics of protein conformational equilibria occurring on the &#181;s-ms timescale. Here, the study presents detailed protocols for acquisition and analysis of a&#160;15N relaxation dispersion experiment. As an example, the pipeline for the analysis of the &#181;s-ms dynamics in the C-terminal domain of bacteria Enzyme I is shown.

15N CPMG Relaxation Dispersion for the Investigation of Protein Conformational Dynamics on the  &#181;s-ms Timescale

Here, a detailed description of the protocol implemented in the laboratory for acquisition and analysis of 15N relaxation dispersion profiles by solution NMR spectroscopy is provided.

Protein conformational dynamics play fundamental roles in regulation of enzymatic catalysis, ligand binding, allostery, and signaling, which are important ...

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...