<meta charset="utf8"></head><body>Monitoramento da GTPase Atlastin em diferentes estados de carga de nucleotídeos

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crossover+conformational+shift+of+the+GTPase+atlastin+provides+the+energy+driving+ER+fusion.">The crossover conformational shift of the GTPase atlastin provides the energy driving ER fusion.</a> J Cell Biol. 216 (5), 1321-1335 (2017).</li><li>Yan, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+the+yeast+dynamin-like+GTPase+sey1p+provide+insight+into+homotypic+er+fusion.">Structures of the yeast dynamin-like GTPase sey1p provide insight into homotypic er fusion.</a> J Cell Biol. 210 (6), 961-972 (2015).</li><li>Rizo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+underlying+neurotransmitter+release.">Molecular mechanisms underlying neurotransmitter release.</a> Annu Rev Biophys. 51, 377-408 (2022).</li><li>Gao, S., Hu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+fusion:+The+machineries+in+and+out.">Mitochondrial fusion: The machineries in and out.</a> Trends Cell Biol. 31 (1), 62-74 (2021).</li><li>Kelly, C. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR-mediated+beta-arrestin+activation+deconvoluted+with+single-molecule+precision.">GPCR-mediated beta-arrestin activation deconvoluted with single-molecule precision.</a> Cell. 185 (10), 1661-1675.e16 (2022).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+DNA+translocation+underlying+chromatin+remodelling+by+snf2.">Mechanism of DNA translocation underlying chromatin remodelling by snf2.</a> Nature. 567 (7748), 409-413 (2019).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+dynamics+of+dynamin-like+MXA+revealed+by+single-molecule+fret.">Conformational dynamics of dynamin-like MXA revealed by single-molecule fret.</a> Nat Commun. 8, 15744 (2017).</li><li>Shi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+mechanism+of+atlastin-mediated+homotypic+membrane+fusion+at+the+single-molecule+level.">Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level.</a> Nat Commun. 15 (1), 2488 (2024).</li><li>Guelly, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+high-throughput+sequencing+identifies+mutations+in+atlastin-1+as+a+cause+of+hereditary+sensory+neuropathy+type+i.">Targeted high-throughput sequencing identifies mutations in atlastin-1 as a cause of hereditary sensory neuropathy type i.</a> Am J Hum Genet. 88 (1), 99-105 (2011).</li><li>Montagna, A., Vajente, N., Pendin, D., Daga, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+CRISPR/Cas9+induced+atlastin+pathological+mutations+in+drosophila.">In vivo analysis of CRISPR/Cas9 induced atlastin pathological mutations in drosophila.</a> Front Neurosci. 14, 547746 (2020).</li><li>Ulengin, I., Park, J. 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<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 K Centrifugal Filters</td>
			<td>Amicon</td>
			<td>UFC901096</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Aminopropyltriethoxysilane (APTES)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td></td>
		</tr>
		<tr>
			<td>45 Ti rotor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>532-nm laser</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>640-nm laser</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7 K MWCO spin desalting columns</td>
			<td>Thermo Scientific</td>
			<td>89882</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5-triphosphate disodium salt (ATP)</td>
			<td>Roche</td>
			<td>11140965001</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzoic acid</td>
			<td>Sigma-Aldrich</td>
			<td>242381</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-PEG-SVA-5000</td>
			<td>Laysan Bio</td>
			<td>170-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti His antibody</td>
			<td>Bioss Antibodies</td>
			<td>bs-0287R-bio</td>
			<td></td>
		</tr>
		<tr>
			<td>d-biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA-free protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>IX897</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#8242;-diphosphate sodium salt (GDP)</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;G7127</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-O-[gamma-thio]triphosphate (GTP&#947;S)</td>
			<td>Roche</td>
			<td>10220647001</td>
			<td></td>
		</tr>
		<tr>
			<td>High numerical aperture oil immersion objective</td>
			<td>Nikon</td>
			<td></td>
			<td>Nikon, 100x, N.A. 1.49, oil immersion</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>https://imagej.net/ij/</td>
		</tr>
		<tr>
			<td>Isopropyl-&#914;-D-Thiogalactoside</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td></td>
		</tr>
		<tr>
			<td>LD555-MAL</td>
			<td>Lumidyne</td>
			<td>4</td>
			<td></td>
		</tr>
		<tr>
			<td>LD655-MAL</td>
			<td>Lumidyne</td>
			<td>10</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamic acid potassium</td>
			<td>Sigma-Aldrich</td>
			<td>G1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium acetate tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M0631</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>The MathWorks, Natick, MA</td>
			<td></td>
			<td>https://www.mathworks.com/</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Fisherbrand</td>
			<td>18834</td>
			<td>&#160;(24 mm &#215; 60 mm)</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td></td>
			<td></td>
			<td>Customized, (75 mm &#215; 26 mm &#215; 1 mm) with 6 pairs of 1.2 mm diameter through holes</td>
		</tr>
		<tr>
			<td>mPEG-SVA-5000</td>
			<td>Laysan Bio</td>
			<td>170-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni Sepharose 6 Fast Flow</td>
			<td>Cytiva</td>
			<td>17531802</td>
			<td></td>
		</tr>
		<tr>
			<td>Origin</td>
			<td>Origin software</td>
			<td></td>
			<td>https://www.originlab.com/</td>
		</tr>
		<tr>
			<td>Polystyrene particles</td>
			<td>QDSphere</td>
			<td>AG1265</td>
			<td></td>
		</tr>
		<tr>
			<td>Protocatechuate 3,4-dioxygenase</td>
			<td>Sigma-Aldrich</td>
			<td>P8279</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Sangon Biotech</td>
			<td>A610492</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase (10/300 GL)</td>
			<td>Cytiva</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>TIRF microscope</td>
			<td>Nikon</td>
			<td>Ti2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td>
			<td>Thermo</td>
			<td>75259</td>
			<td></td>
		</tr>
	</tbody>
</table>

single-molecule fret imaging for observing the conformational dynamics of dynamin-like gtpase atlastin

<meta charset="utf8"></head><body>Imagem FRET de molécula única para observar a dinâmica conformacional da GTPase Atlastin semelhante à dinamina

Imagem FRET de molécula única para observar a dinâmica conformacional da GTPase Atlastin semelhante à dinamina

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver ...

single-molecule-fret-imaging-for-observing-conformational-dynamics

Research

JoVE Journal

Biochemistry

Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

297 visualizações.  Nankai University. As funções das proteínas da superfamília da dinamina dependem de mudanças conformacionais associadas à hidrólise do GTP. Um sistema é descrito usando a técnica FRET de molécula única (smFRET) para monitorar a dinâmica conformacional da GTPase aplastina semelhante à dinamina em diferentes estados de carga de nucleotídeos.

Vídeo: Imagem FRET de molécula única para observar a dinâmica conformacional da GTPase Atlastin semelhante à dinamina

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.

Informação estrutural de uma única molécula de FRET experimentos usando a Nano-posicionamento sistema rápido

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

Bioquímica

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.

FRET de alta precisão em nível de molécula única para determinação de estrutura de biomoléculas

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.

Usando três cores único-molécula FRET para estudar a correlação de interações da proteína

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.

Quantificação de Proteome-largo de rotulagem homogeneidade a nível da molécula

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

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Produção de conjuntos de motor de dynein e de Kinesin em nanostructures do ADN origami para a única observação da molécula

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Um ensaio de imagem de molécula única in vitro para a análise de cinética de tradução dependente da tampa

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

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.

Fazendo medições de FRET de molécula única precisas e precisas usando o smfBox de código aberto

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.

15 N CPMG Relaxation Dispersão para a Investigação da Dinâmica Conformacional de Proteínas na escala de tempo μs-ms

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

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

Imagem de molécula única de condensados EWS-FLI1 montados no DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

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.

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.