<meta charset="utf8"></head><body>监测不同核苷酸负载状态下的 GTP 酶 Atlastin

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamin+superfamily.">The dynamin superfamily.</a> Curr Biol. 28 (8), R411-R416 (2018).</li><li>Bryce, S., 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=Human+atlastin-3+is+a+constitutive+ER+membrane+fusion+catalyst.">Human atlastin-3 is a constitutive ER membrane fusion catalyst.</a> J Cell Biol. 222 (7), e202211021 (2023).</li><li>Crosby, D., 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=Reconstitution+of+human+atlastin+fusion+activity+reveals+autoinhibition+by+the+C+terminus.">Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus.</a> J Cell Biol. 221 (2), e202107070 (2022).</li><li>Hu, J., 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=A+class+of+dynamin-like+GTPases+involved+in+the+generation+of+the+tubular+er+network.">A class of dynamin-like GTPases involved in the generation of the tubular er network.</a> Cell. 138 (3), 549-561 (2009).</li><li>Jang, E., 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=Human+atlastins+are+sufficient+to+drive+the+fusion+of+liposomes+with+a+physiological+lipid+composition.">Human atlastins are sufficient to drive the fusion of liposomes with a physiological lipid composition.</a> J Cell Biol. 222 (4), e202109090 (2023).</li><li>Liu, X., 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=Atlastin-1+regulates+morphology+and+function+of+endoplasmic+reticulum+in+dendrites.">Atlastin-1 regulates morphology and function of endoplasmic reticulum in dendrites.</a> Nat Commun. 10 (1), 568 (2019).</li><li>Orso, G., 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=Homotypic+fusion+of+ER+membranes+requires+the+dynamin-like+gtpase+atlastin.">Homotypic fusion of ER membranes requires the dynamin-like gtpase atlastin.</a> Nature. 460 (7258), 978-983 (2009).</li><li>Rismanchi, N., Soderblom, C., Stadler, J., Zhu, P. P., Blackstone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlastin+GTPases+are+required+for+Golgi+apparatus+and+er+morphogenesis.">Atlastin GTPases are required for Golgi apparatus and er morphogenesis.</a> Hum Mol Genet. 17 (11), 1591-1604 (2008).</li><li>Faust, J. E., 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=The+atlastin+C-terminal+tail+is+an+amphipathic+helix+that+perturbs+the+bilayer+structure+during+endoplasmic+reticulum+homotypic+fusion.">The atlastin C-terminal tail is an amphipathic helix that perturbs the bilayer structure during endoplasmic reticulum homotypic fusion.</a> J Biol Chem. 290 (8), 4772-4783 (2015).</li><li>Liu, T. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+fusion+by+the+GTPase+atlastin+requires+a+conserved+c-terminal+cytoplasmic+tail+and+dimerization+through+the+middle+domain.">Membrane fusion by the GTPase atlastin requires a conserved c-terminal cytoplasmic tail and dimerization through the middle domain.</a> Proc Natl Acad Sci U S A. 108 (27), 11133-11138 (2011).</li><li>Bian, X., 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+atlastin+GTpase+provide+insight+into+homotypic+fusion+of+endoplasmic+reticulum+membranes.">Structures of the atlastin GTpase provide insight into homotypic fusion of endoplasmic reticulum membranes.</a> Proc Natl Acad Sci U S A. 108 (10), 3976-3981 (2011).</li><li>Byrnes, L. J., 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=Structural+basis+for+conformational+switching+and+GTP+loading+of+the+large+g+protein+atlastin.">Structural basis for conformational switching and GTP loading of the large g protein atlastin.</a> EMBO J. 32 (3), 369-384 (2013).</li><li>Byrnes, L. J., Sondermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+nucleotide-dependent+dimerization+of+the+large+g+protein+atlastin-1/spg3a.">Structural basis for the nucleotide-dependent dimerization of the large g protein atlastin-1/spg3a.</a> Proc Natl Acad Sci U S A. 108 (6), 2216-2221 (2011).</li><li>Morin-Leisk, J., 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=An+intramolecular+salt+bridge+drives+the+soluble+domain+of+gtp-bound+atlastin+into+the+postfusion+conformation.">An intramolecular salt bridge drives the soluble domain of gtp-bound atlastin into the postfusion conformation.</a> J Cell Biol. 195 (4), 605-615 (2011).</li><li>Pendin, D., 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=GTP-dependent+packing+of+a+three-helix+bundle+is+required+for+atlastin-mediated+fusion.">GTP-dependent packing of a three-helix bundle is required for atlastin-mediated fusion.</a> Proc Natl Acad Sci U S A. 108 (39), 16283-16288 (2011).</li><li>Winsor, J., Hackney, D. D., Lee, T. 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. M., Byrnes, L. J., Neela, N., Sondermann, H., O'donnell, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hypervariable+region+of+atlastin-1+is+a+site+for+intrinsic+and+extrinsic+regulation.">The hypervariable region of atlastin-1 is a site for intrinsic and extrinsic regulation.</a> J Cell Biol. 220 (11), e202104128 (2021).</li><li>Asher, W. 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. J., Lee, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ER+network+formation+and+membrane+fusion+by+atlastin1/spg3a+disease+variants.">ER network formation and membrane fusion by atlastin1/spg3a disease variants.</a> Mol Biol Cell. 26 (9), 1616-1628 (2015).</li><li>Zhao, X., 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=Mutations+in+a+newly+identified+GTPase+gene+cause+autosomal+dominant+hereditary+spastic+paraplegia.">Mutations in a newly identified GTPase gene cause autosomal dominant hereditary spastic paraplegia.</a> Nat Genet. 29 (3), 326-331 (2001).</li></ol>

<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>用于观察动力蛋白样 GTP 酶弹性蛋白构象动力学的单分子 FRET 成像

用于观察动力蛋白样 GTP 酶弹性蛋白构象动力学的单分子 FRET 成像

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

293 次观看.  Nankai University. dynamin 超家族蛋白的功能取决于与 GTP 水解偶联的构象变化。使用单分子 FRET （smFRET） 技术描述了一个系统，用于监测不同核苷酸负载状态下动力蛋白样 GTP 酶弹性蛋白的构象动力学。

视频: 用于观察动力蛋白样 GTP 酶弹性蛋白构象动力学的单分子 FRET 成像

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.

从单分子结构信息FRET实验使用快速纳米定位系统

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

科研

生物化学

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.

单分子水平的高精度FRET用于生物分子结构测定

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.

应用三色单分子焦虑研究蛋白质相互作用的相关性

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

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

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.

单分子水平标记均匀性的全蛋白质定量

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.

生产用于单分子观察的DNA折纸纳米结构的Dynein和Kinesin电机组合

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.

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

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.

使用开源 smfBox 进行精确和准确的单分子 FRET 测量

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.

15N CPMG 放松分散，用于研究 μs-ms 时间尺度上的蛋白质符合性动力学

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.

EWS-FLI1凝聚物在DNA上组装的单分子成像

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 ...