Name: using three-color single-molecule fret to study the correlation of protein interactions
Uploaded: 2018-01-30T14:00:00
Description: Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

Эта работа финансируется немецкого фонда научных исследований (INST 39/969-1) и Европейский исследовательский совет через соглашение грантов ЕИС n. 681891.

1. Установка и предпосылки
<ol><li>Выполните измерения многоцветные smFRET Призма типа TIRFM. Описание установки двух цветные как Зевс публикации дается ссылка12.<ol>
<li>Постройте TIRFM многоцветные. Генеральный план подробно в 9.</li>
		<li>Использование переключаемый, диодной ...

<ol>
	<li>Nooren, I. M. A., Thornton, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+protein-protein+interactions.">Diversity of protein-protein interactions.</a> EMBO J. 22 (14), 3486-3492 (2003).</li><li>Marsh, J. A., Teichmann, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+dynamics,+assembly,+and+evolution+of+protein+complexes.">Structure, dynamics, assembly, and evolution of protein complexes.</a> Annu Rev Biochem. 84, 551-575 (2015).</li><li>Henzler-Wildman, K., Kern, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+personalities+of+proteins.">Dynamic personalities of proteins.</a> Nature. 450 (7172), 964-972 (2007).</li><li>Hohng, S., Joo, C., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Three-Color+FRET.">Single-Molecule Three-Color FRET.</a> Biophys J. 87 (2), 1328-1337 (2004).</li><li>Person, B., Stein, I. H., Steinhauer, C., Vogelsang, J., Tinnefeld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlated+movement+and+bending+of+nucleic+acid+structures+visualized+by+multicolor+single-molecule+spectroscopy.">Correlated movement and bending of nucleic acid structures visualized by multicolor single-molecule spectroscopy.</a> ChemPhysChem. 10 (9-10), 1455-1460 (2009).</li><li>Lee, J., Lee, S., Ragunathan, K., Joo, C., Ha, T., Hohng, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+four-color+FRET.">Single-molecule four-color FRET.</a> Angew Chem Int Ed. 49 (51), 9922-9925 (2010).</li><li>Ratzke, C., Berkemeier, F., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+90's+mechanochemical+cycle+is+dominated+by+thermal+fluctuations.">Heat shock protein 90's mechanochemical cycle is dominated by thermal fluctuations.</a> Proc Natl Acad Sci U S A. 109 (1), 161-166 (2012).</li><li>Ratzke, C., Hellenkamp, B., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four-colour+FRET+reveals+directionality+in+the+Hsp90+multicomponent+machinery.">Four-colour FRET reveals directionality in the Hsp90 multicomponent machinery.</a> Nat Commun. 5, 4192 (2014).</li><li>G&#246;tz, M., Wortmann, P., Schmid, S., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multicolor+Single-Molecule+FRET+Approach+to+Study+Protein+Dynamics+and+Interactions+Simultaneously.">A Multicolor Single-Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously.</a> Methods Enzymol. 581, 487-516 (2016).</li><li>Yengo, C. M., Berger, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+anisotropy+and+resonance+energy+transfer:+Powerful+tools+for+measuring+real+time+protein+dynamics+in+a+physiological+environment.">Fluorescence anisotropy and resonance energy transfer: Powerful tools for measuring real time protein dynamics in a physiological environment.</a> Curr Opin Pharmacol. 10 (6), 731-737 (2010).</li><li>Wortmann,P , ., Götz M, ., Hugel T , . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Nucleotide+Binding+in+Hsp90+and+Its+Regulation+by+Aha1.">Cooperative Nucleotide Binding in Hsp90 and Its Regulation by Aha1.</a> Biophys J. 113, 1711-1718 (2017).</li><li>D&#246;rfler, T., Eilert, T., R&#246;cker, C., Nagy, J., Michaelis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Information+from+Single-molecule+FRET+Experiments+Using+the+Fast+Nano-positioning+System.">Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System.</a> J Vis Exp. (120), e54782 (2017).</li><li>Stephanopoulos, N., Francis, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+effective+protein+bioconjugation+strategy.">Choosing an effective protein bioconjugation strategy.</a> Nature chemical biology. 7 (12), 876-884 (2011).</li><li>S&#225;nchez-Rico, C., Voith von Voithenberg, L., Warner, L., Lamb, D. C., Sattler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Fluorophore+Attachment+on+Protein+Conformation+and+Dynamics+Studied+by+spFRET+and+NMR+Spectroscopy.">Effects of Fluorophore Attachment on Protein Conformation and Dynamics Studied by spFRET and NMR Spectroscopy.</a> Chemistry (Weinheim an der Bergstrasse, Germany). , (2017).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nat Methods. 5 (6), 507-516 (2008).</li><li>Lee, N. K., 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=Three-color+alternating-laser+excitation+of+single+molecules:+monitoring+multiple+interactions+and+distances.">Three-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances.</a> Biophys J. 92 (1), 303-312 (2007).</li><li>Kapanidis, A. N., Lee, N. K., Laurence, T. A., Doose, S., Margeat, E., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-aided+molecule+sorting:+analysis+of+structure+and+interactions+by+alternating-laser+excitation+of+single+molecules.">Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules.</a> Proc Natl Acad Sci U S A. 101 (24), 8936-8941 (2004).</li><li>Hohlbein, J., Craggs, T. D., Cordes, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternating-laser+excitation:+single-molecule+FRET+and+beyond.">Alternating-laser excitation: single-molecule FRET and beyond.</a> Chem Soc Rev. 43 (4), 1156-1171 (2014).</li><li>Hellenkamp, B., Wortmann, P., Kandzia, F., Zacharias, M., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidomain+structure+and+correlated+dynamics+determined+by+self-consistent+FRET+networks.">Multidomain structure and correlated dynamics determined by self-consistent FRET networks.</a> Nat Methods. 14, 174-180 (2017).</li><li>Rabiner, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tutorial+on+hidden+Markov+models+and+selected+applications+in+speech+recognition.">A tutorial on hidden Markov models and selected applications in speech recognition.</a> Proc IEEE. 77 (2), 257-286 (1989).</li><li>Fink, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Markov Models for Pattern Recognition. , (2014).</li><li>Giudici, P., Ryden, T., Vandekerkhove, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Likelihood-Ratio+Tests+for+Hidden+Markov+Models.">Likelihood-Ratio Tests for Hidden Markov Models.</a> Biometrics. 56 (3), 742-747 (2000).</li><li>Visser, I., Raijmakers, M. E. J., Molenaar, P. C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confidence+intervals+for+hidden+Markov+model+parameters.">Confidence intervals for hidden Markov model parameters.</a> Br J Math Stat Psychol. 53 (2), 317-327 (2000).</li><li>McKinney, S. A., Joo, C., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Single-Molecule+FRET+Trajectories+Using+Hidden+Markov+Modeling.">Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling.</a> Biophys J. 91 (5), 1941-1951 (2006).</li><li>Bronson, J. E., Fei, J., Hofman, J. M., Gonzalez, R. L., Wiggins, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+Rates+and+States+from+Biophysical+Time+Series:+A+Bayesian+Approach+to+Model+Selection+and+Single-Molecule+FRET+Data.">Learning Rates and States from Biophysical Time Series: A Bayesian Approach to Model Selection and Single-Molecule FRET Data.</a> Biophys J. 97 (12), 3196-3205 (2009).</li><li>Greenfeld, M., Pavlichin, D. S., Mabuchi, H., Herschlag, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Molecule+Analysis+Research+Tool+(SMART):+an+integrated+approach+for+analyzing+single+molecule+data.">Single Molecule Analysis Research Tool (SMART): an integrated approach for analyzing single molecule data.</a> PLoS ONE. 7 (2), e30024 (2012).</li><li>Schmid, S., G&#246;tz, M., Hugel, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Molecule+Analysis+beyond+Dwell+Times:+Demonstration+and+Assessment+in+and+out+of+Equilibrium.">Single-Molecule Analysis beyond Dwell Times: Demonstration and Assessment in and out of Equilibrium.</a> Biophys J. 111 (7), 1375-1384 (2016).</li><li>Taipale, M., Jarosz, D. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+at+the+hub+of+protein+homeostasis:+emerging+mechanistic+insights.">HSP90 at the hub of protein homeostasis: emerging mechanistic insights.</a> Nat Rev Mol Cell Biol. 11 (7), 515-528 (2010).</li><li>Trepel, J., Mollapour, M., Giaccone, G., Neckers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+dynamic+HSP90+complex+in+cancer.">Targeting the dynamic HSP90 complex in cancer.</a> Nat Rev Cancer. 10 (8), 537-549 (2010).</li><li>Wayne, N., Bolon, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimerization+of+Hsp90+is+required+for+in+vivo+function.+Design+and+analysis+of+monomers+and+dimers.">Dimerization of Hsp90 is required for in vivo function. Design and analysis of monomers and dimers.</a> J Biol Chem. 282 (48), 35386-35395 (2007).</li><li>Ali, M. M. U., 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=Crystal+structure+of+an+Hsp90-nucleotide-p23/Sba1+closed+chaperone+complex.">Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex.</a> Nature. 440 (7087), 1013-1017 (2006).</li><li>Southworth, D. R., Agard, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-dependent+ensembles+of+conserved+conformational+states+define+the+Hsp90+chaperone+ATPase+cycle.">Species-dependent ensembles of conserved conformational states define the Hsp90 chaperone ATPase cycle.</a> Mol Cell. 32 (5), 631-640 (2008).</li><li>Blanchard, S. C., Kim, H. D., Gonzalez, R. L., Puglisi, J. D., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=tRNA+dynamics+on+the+ribosome+during+translation.">tRNA dynamics on the ribosome during translation.</a> Proc Natl Acad Sci U S A. 101 (35), 12893-12898 (2004).</li><li>Aitken, C. E., Marshall, R. A., Puglisi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Oxygen+Scavenging+System+for+Improvement+of+Dye+Stability+in+Single-Molecule+Fluorescence+Experiments.">An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments.</a> Biophys J. 94 (5), 1826-1835 (2008).</li><li>Swoboda, 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=Enzymatic+oxygen+scavenging+for+photostability+without+pH+drop+in+single-molecule+experiments.">Enzymatic oxygen scavenging for photostability without pH drop in single-molecule experiments.</a> ACS Nano. 6 (7), 6364-6369 (2012).</li><li>Rognoni, L., Stigler, J., Pelz, B., Yl&#228;nne, J., Rief, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+force+sensing+of+filamin+revealed+in+single-molecule+experiments.">Dynamic force sensing of filamin revealed in single-molecule experiments.</a> Proc Natl Acad Sci U S A. 109 (48), 19679-19684 (2012).</li><li>Okumus, B., Wilson, T. J., Lilley, D. M. J., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vesicle+encapsulation+studies+reveal+that+single+molecule+ribozyme+heterogeneities+are+intrinsic.">Vesicle encapsulation studies reveal that single molecule ribozyme heterogeneities are intrinsic.</a> Biophys J. 87 (4), 2798-2806 (2004).</li><li>Boukobza, E., Sonnenfeld, A., Haran, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+in+Surface-Tethered+Lipid+Vesicles+as+a+New+Tool+for+Single+Biomolecule+Spectroscopy.">Immobilization in Surface-Tethered Lipid Vesicles as a New Tool for Single Biomolecule Spectroscopy.</a> J Phys Chem B. 105 (48), 12165-12170 (2001).</li><li>Levene, M. J., Korlach, J., Turner, S. W., Foquet, M., Craighead, H. G., Webb, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zero-mode+waveguides+for+single-molecule+analysis+at+high+concentrations.">Zero-mode waveguides for single-molecule analysis at high concentrations.</a> Science. 299 (5607), 682-686 (2003).</li><li>Panaretou, 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=ATP+binding+and+hydrolysis+are+essential+to+the+function+of+the+Hsp90+molecular+chaperone+in+vivo.">ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.</a> EMBO J. 17 (16), 4829-4836 (1998).</li></ol>

Мы представляем экспериментальная процедура получения трехцветных smFRET данных для сложных белков системы и пошаговое описание анализа этих измерений. Этот подход предоставляет уникальную возможность непосредственно оценить взаимосвязь между несколькими взаимодействия сайтов или к...

<table border="0" cellpadding="0" cellspacing="0" width="1301">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Setup</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td style="width:347px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">vibration-damped optical table&#160;&#160;</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td style="width:167px;">RS2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 473nm LX 75mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1185052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 532nm LS 50mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1261779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 594nm LS 60mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1233470</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">OBIS 637nm LX 140mW LASER</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1196625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">laser control unit</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;">1234465</td>
			<td style="width:347px;">Scientific Remote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aspheric telescope lenses</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>d=25.4mm, f=50mm and f=100mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 473/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 532/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZET 594/10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CF ex4</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td>FL635-10</td>
			<td>cleanup filter excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ594RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">570DCXR</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM ex3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">ZQ491RDC</td>
			<td>dichroic mirror excitation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AOTFnC-Vis</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">&#955;/4 plate</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;">AQWP05M-600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">CFI Apo TIRF 100x</td>
			<td style="width:445px;">Nikon Instruments Inc, Melville, NY, USA</td>
			<td style="width:167px;"></td>
			<td>high-NA objective</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo focus positioner MIPOS 250 CAP</td>
			<td style="width:445px;">piezosystem jena GmbH, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td>Piezo Controller NV 40/1 CLE</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">piezo stepper</td>
			<td style="width:445px;">Newport, Irvine, CA, USA</td>
			<td>PZA12</td>
			<td>PZC200-KT NanoPZ Actuator Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">achromatic aspheric lenses</td>
			<td style="width:445px;">Qioptiq Photonics GmbH &#38; Co. KG, G&#246;ttingen, Germany</td>
			<td style="width:167px;">G322-304-000</td>
			<td>d=50mm, f=200mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">adjustable optical slit</td>
			<td style="width:445px;">Owis GmbH, Staufen i. Br., Germany</td>
			<td>27.160.1212</td>
			<td>max. aperture 12 x 12 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">T 600 LPXR</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">H 560 LPXR superflat</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">DM det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">HC BS R635</td>
			<td>dichroic mirror detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det1</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">525/40 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det2</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">586/20 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det3</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">631/36 BrightLine HC</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">BP det4</td>
			<td style="width:445px;">AHF analysentechnik AG, T&#252;bingen, Germany</td>
			<td style="width:167px;">700/75 ET Bandpass</td>
			<td>bandpass filter detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">optical shutters detection</td>
			<td style="width:445px;">Vincent Associates, Rochester, NY, USA</td>
			<td style="width:167px;">Uniblitz VS25S2T0&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">EMCCD iXon Ultra 897</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">digital I/O card, PCIe-6535</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">syringe pump</td>
			<td style="width:445px;">Harvard Apparatus, Holliston, MA, USA</td>
			<td style="width:167px;">PHD22/2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Flow chamber</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">quartz slides</td>
			<td style="width:445px;">G. Finkenbeiner Inc, Waltham, MA, USA</td>
			<td style="width:167px;"></td>
			<td>Spectrosil2000, h=3mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">TEGADERM film</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">1626W</td>
			<td>10 x 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">spray adhesive</td>
			<td style="width:445px;">3M Deutschland GmbH, Neuss, Germany</td>
			<td style="width:167px;">Photo Mount 050777</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">glycerol</td>
			<td style="width:445px;">Carl Zeiss AG, Oberkochen, Germany</td>
			<td style="width:167px;">Immersol G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">immersion oil</td>
			<td style="width:445px;">OLYMPUS EUROPA SE &#38; CO. KG, Hamburg, Germany</td>
			<td>IMMOIL-F30CC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">prism</td>
			<td style="width:445px;">Vogelsberger Quarzglastechnik GmbH, Hauzenberg, Germany</td>
			<td style="width:167px;"></td>
			<td>Suprasil1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">aluminium prism holder</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>custom built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">hollow setscrews</td>
			<td style="width:445px;">Thorlabs Inc, Newton, New Jersey, USA</td>
			<td style="width:167px;"></td>
			<td>with custom drilling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Tygon S3 E-3603 tubing</td>
			<td style="width:445px;">neoLab Migge GmbH, Heidelberg, Germany</td>
			<td style="width:167px;">2-4450</td>
			<td>ACF00001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">PTFE tubing</td>
			<td style="width:445px;">Bohlender GmbH, Gr&#252;nsfeld, Germany</td>
			<td style="width:167px;">S1810-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sample</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">yeast Hsp90 D61C, Q385C_biotin</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td>UniProt ID P02829</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Maleimide derivatives of Atto488, Atto550</td>
			<td style="width:445px;">ATTO-TEC GmbH, Siegen, Germany</td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">AMP-PNP*</td>
			<td style="width:445px;">Jena Bioscience, Jena, Germany</td>
			<td style="width:167px;"></td>
			<td style="width:347px;">&#947;-[(6-Aminohexyl)-imido]-AMP-PNP-Atto647N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Fluospheres</td>
			<td style="width:445px;">Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td style="width:167px;">F8764</td>
			<td>amine-modified, 0.2 &#956;m, yellow-green fluorescent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Name</td>
			<td style="width:445px;">Company</td>
			<td style="width:167px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Software</td>
			<td style="width:445px;"></td>
			<td style="width:167px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Andor Solis</td>
			<td style="width:445px;">Andor Technology Ltd, Belfast, Northern Ireland</td>
			<td style="width:167px;"></td>
			<td>version 4.30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">LabVIEW</td>
			<td style="width:445px;">National Instruments, Austin, Texas, USA</td>
			<td style="width:167px;"></td>
			<td>version 2012, 32bit; misc. hardware control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">MDS control software</td>
			<td style="width:445px;">AA Opto-Electronic, Orsay, France</td>
			<td style="width:167px;"></td>
			<td>version 2.03a</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Coherent Connection</td>
			<td style="width:445px;">Coherent Inc, Santa Clara, CA, USA</td>
			<td style="width:167px;"></td>
			<td>version 3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Igor Pro</td>
			<td style="width:445px;">WaveMetrics Inc, Portland, OR, USA</td>
			<td style="width:167px;"></td>
			<td>version 6.37</td>
		</tr>
	</tbody>
</table>

using three-color single-molecule fret to study the correlation of protein interactions

Одноместный молекула Фёрстер резонанс передачи энергии (smFRET) стала широко используемый биофизический метод для изучения динамики биомолекул. Для многих молекулярных машин в клетки белки должны действовать вместе с взаимодействия партнеров в функциональных цикла для выполнения их задачи. Расширение два цвета многоцветные smFRET позволяет одновременно исследовать более чем одной взаимодействия или конформационные изменения. Это не только добавляет новое измерение к smFRET эксперименты, но он также предлагает уникальную возможность непосредственно изучить последовательность событий и Обнаружение коррелированных взаимодействия при использовании иммобилизованных образца и полного внутреннего отражения флуоресценции Микроскоп (TIRFM). Таким образом многоцветные smFRET является универсальным инструментом для изучения биомолекулярных комплексов в количественном выражении и ранее недостижимое подробно.
Здесь мы продемонстрируем для преодоления особых проблем многоцветные smFRET экспериментов на белки. Мы представляем подробные протоколы для получения данных и извлечения кинетическая информации. Это включает в себя критерии отбора трассировки, разделение государства и восстановления государства траекторий от шумных данных с использованием 3D ансамбль скрытой марковской модели (HMM). По сравнению с другими методами, кинетическая информация не восстанавливается от гистограммы времени останавливаться, но непосредственно от Хм. Рамки максимального правдоподобия позволяет нам критически оценивать кинетическая модель и для обеспечения значимого неопределенности для ставок.
Применяя метод белка теплового шока 90 (Hsp90), мы в состоянии отделить нуклеотида привязки и глобальные конформационные изменения белка. Это позволяет нам непосредственно наблюдать кооперативность между двумя нуклеотидов привязки очагов Hsp90 димер.

Многоцветные smFRET измерения позволяют прямое обнаружение корреляции между двумя или более сайтами различных взаимодействия. Это делает технику уникальный расследовать многокомпонентных систем, таких как белковых комплексов. Мы сосредоточены на презентации трехцве?...

Watch this Scientific Journal Video about Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions at JoVE.com

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

С использованием трехцветной сингл молекула лад для изучения корреляции взаимодействий протеина

Здесь мы представляем протокол для получения трехцветных smFRET данных и их анализ с 3D ансамбль скрытой марковской модели. С этим подходом ученые могут извлекать кинетическая информацию из сложных белковых систем, включая кооперативность или коррелированные взаимодействий.

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

Research

JoVE Journal

Biochemistry

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Анизотропию, как флуоресценция инструмента для изучения белок-белковых взаимодействий

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

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

Структурная информация от одной молекулы FRET Эксперименты с помощью быстрого нано-системы позиционирования

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

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Используя строительные леса, липосом Развести липидами проксимальный белок-белковых взаимодействий<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

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

Высокая точность FRET на уровне одиночной молекулы для определения структуры биомолекулы

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

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Биотинилированным клеток-проникая пептиды для изучения внутриклеточный белок белковых взаимодействий

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Ковалентной иммобилизации белков для одной молекулы силой спектроскопии

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

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

OaAEP1-опосредованный ферментативный синтез и иммобилизация полимеризованного белка для одномолекулярной силовой спектроскопии

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

In Vivo Hydroxyl Radical Protein Footprinting for the Study of Protein Interactions in Caenorhabditis elegans

В Vivo Hydroxyl радикальный белок следдля изучения взаимодействия белков в Caenorhabditis elegans

Fast oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting (HRPF) method used to study protein structure, protein-ligand interactions, ...

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

Проведение точных и точных одномолекулярных измерений FRET с помощью smfBox с открытым исходным кодом

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