Name: measuring protein binding to f-actin by co-sedimentation
Uploaded: 2017-05-18T12:30:00.000+00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

Este trabajo fue apoyado por el National Institutes of Health Grant HL127711 a AVK.

<ol>
	<li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+and+Actin-Binding+Proteins.">Actin and Actin-Binding Proteins.</a> Cold Spring Harb Perspect Biol. 8 (8), (2016).</li><li>Hansen, M. D., Kwiatkowski, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+actin+dynamics+by+allosteric+regulation+of+actin+binding+proteins.">Control of actin dynamics by allosteric regulation of actin binding proteins.</a> Int Rev Cell Mol Biol. 303, 1-25 (2013).</li><li>Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin-binding+proteins:+the+long+road+to+understanding+the+dynamic+landscape+of+cellular+actin+networks.">Actin-binding proteins: the long road to understanding the dynamic landscape of cellular actin networks.</a> Mol Biol Cell. 27 (16), 2519-2522 (2016).</li><li>Mullins, R. D., Hansen, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+studies+of+actin+filament+and+network+dynamics.">In vitro studies of actin filament and network dynamics.</a> Curr Opin Cell Biol. 25 (1), 6-13 (2013).</li><li>Miller, P. W., 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=Danio+rerio+alphaE-catenin+is+a+monomeric+F-actin+binding+protein+with+distinct+properties+from+Mus+musculus+alphaE-catenin.">Danio rerio alphaE-catenin is a monomeric F-actin binding protein with distinct properties from Mus musculus alphaE-catenin.</a> J Biol Chem. 288 (31), 22324-22332 (2013).</li><li>Wickline, E. 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=alphaT-Catenin+Is+a+Constitutive+Actin-binding+alpha-Catenin+That+Directly+Couples+the+Cadherin.Catenin+Complex+to+Actin+Filaments.">alphaT-Catenin Is a Constitutive Actin-binding alpha-Catenin That Directly Couples the Cadherin.Catenin Complex to Actin Filaments.</a> J Biol Chem. 291 (30), 15687-15699 (2016).</li><li>Simpson, R. J., Adams, P. D., Golemis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic methods in protein purification and analysis : a laboratory manual. , (2009).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Dyballa, N., Metzger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+sensitive+colloidal+coomassie+G-250+staining+for+proteins+in+polyacrylamide+gels.">Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels.</a> J Vis Exp. (30), (2009).</li><li>Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+simple+and+informative+binding+assays.">A guide to simple and informative binding assays.</a> Mol Biol Cell. 21 (23), 4061-4067 (2010).</li><li>Hansen, S. 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=alphaE-catenin+actin-binding+domain+alters+actin+filament+conformation+and+regulates+binding+of+nucleation+and+disassembly+factors.">alphaE-catenin actin-binding domain alters actin filament conformation and regulates binding of nucleation and disassembly factors.</a> Mol Biol Cell. 24 (23), 3710-3720 (2013).</li><li>Perrin, B. J., Ervasti, 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=The+actin+gene+family:+function+follows+isoform.">The actin gene family: function follows isoform.</a> Cytoskeleton (Hoboken). 67 (10), 630-634 (2010).</li><li>Tondeleir, D., Vandamme, D., Vandekerckhove, J., Ampe, C., Lambrechts, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+isoform+expression+patterns+during+mammalian+development+and+in+pathology:+insights+from+mouse+models.">Actin isoform expression patterns during mammalian development and in pathology: insights from mouse models.</a> Cell Motil Cytoskeleton. 66 (10), 798-815 (2009).</li><li>Prochniewicz, E., Janson, N., Thomas, D. D., Dela Cruz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+increases+the+torsional+flexibility+and+dynamics+of+actin+filaments.">Cofilin increases the torsional flexibility and dynamics of actin filaments.</a> J Mol Biol. 353 (5), 990-1000 (2005).</li><li>Kuhn, J. R., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+measurements+of+actin+filament+polymerization+by+total+internal+reflection+fluorescence+microscopy.">Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy.</a> Biophys J. 88 (2), 1387-1402 (2005).</li><li>Hansen, S. D., Zuchero, J. B., Mullins, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+actin:+purification+and+single+molecule+assembly+assays.">Cytoplasmic actin: purification and single molecule assembly assays.</a> Methods Mol Biol. 1046, 145-170 (2013).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

El ensayo de co-sedimentación de actina es una técnica sencilla que puede determinar rápidamente si una proteína se une a la F-actina. Con algunas modificaciones, la técnica también puede usarse para medir la afinidad de la interacción. Además de los puntos planteados en el protocolo anterior, los siguientes aspectos deben ser considerados al diseñar, conducir e interpretar el ensayo. Proteína de Interés En el ensayo se puede usar proteína recién preparada o congelada. Si se usa proteína congelada, se recomienda que los resultados se comparen con proteínas frescas (nunca congeladas) para asegurar que la congelación no afecta a la unión de F-actina. G-actina Fuente Muchos experimentos de granulación utilizan G-actina aislada del músculo debido a su abundancia relativa. Hay tres principales isotipos de actina en mamíferos - alfa, beta y gamma - que son notablemente similares (&gt; 90% de la secuencia identiTy). No obstante, existen diferencias funcionales entre los isotipos 12 , 13 . Si es posible, el isotipo G-actina usado en el ensayo de unión debe coincidir con el isotipo in vivo . Por ejemplo, si se prueba una proteína expresada en el músculo esquelético, la alfa-actina es la mejor opción; Si se examina una proteína expresada en fibroblastos, se recomienda la beta-actina. Uso de Phalloidin Dado que la faloidina se une a la actina F, puede interferir o incluso bloquear la unión de algunas proteínas de unión a la actina F ( p. Ej., La faloidina bloquea la cofilina de la unión a los filamentos de actina) 14 . Por lo tanto, la faloidina debe utilizarse con precaución y los resultados en comparación con las muestras no tratados con faloidina cuando sea posible. Fondo alto No es infrecuente que las proteínas sedimenten en ausencia de actina F ( Figura 1A , ninguna muestra de pellet de F-actinaS). Sin embargo, altos niveles de sedimentación de fondo pueden enmascarar la verdadera co-sedimentación de actina y dificultar, si no imposible, determinar si una proteína se une a F-actina o para medir la afinidad de la interacción. La adición de polidicanol al tampón de reacción (paso 4.1) puede reducir significativamente el fondo y es una solución fácil. Si esto no reduce el fondo, el ajuste del tampón de reacción, la concentración de sal y / o la temperatura de incubación pueden ayudar. Curva de encuadernación Para generar una curva de unión, es necesario variar la concentración de la proteína de interés o de la F-actina sobre una serie de reacciones. En la práctica, es más fácil y preferible mantener F-actina a una concentración fija y variar la concentración de la proteína de interés. El mantenimiento de la actina F a una concentración fija ( por ejemplo, 2 μM) en el ensayo de granulación limita la captura no específica a concentraciones más altas de actina F y previeneDespolimerización a concentraciones menores (&lt;0,5 μM) de F-actina. La despolimerización puede evitarse utilizando la faloidina, aunque esto introduce un factor de complicación potencial en el sistema (ver paso 3.3 y anteriores). El mantenimiento de la actina F a una concentración fija también permite comparar (y normalizar) el sedimento de actina F entre muestras e identificar experimentos fallidos ( es decir, donde el sedimento de actina F es altamente variable, evitando el análisis a través de las concentraciones). Por último, el mantenimiento de la actina F a una concentración fija permite determinar si la unión al filamento de actina es cooperativa ( Figura 1C ). Unión saturada Como en todos los experimentos de unión, es crítico que la unión a F-actina esté saturada y que la concentración de proteína más platinas de F-actina ( Figura 1C ). Sin una meseta, no es posible calcular una constante de equilibrio de disociación precisa. Por lo tanto,Es importante planificar cuidadosamente la serie de dilución a ensayar e incluir siempre concentraciones más altas de proteína ( es decir, por lo menos 5- a 10 veces mayor que la Kd esperada). Análisis de encuadernación Para que las constantes de disociación medidas sean concluyentes, el ensayo debe realizarse usando una concentración de actina F que permita que la concentración de sitios de unión en la actina F para la proteína de interés sea mucho menor que la afinidad. Para comprobar si este criterio se cumplió, estimar la concentración de sitios de unión de B max . Por ejemplo, si [F-actina] era 2 μM y Bmax = 0,5, entonces [sitios de unión] ≈ 1 μM. El Kd debe ser al menos 5- a 10 veces mayor que [sitios de unión]. Si el Kd medido es del mismo orden de magnitud que [sitios de unión], entonces es posible que la curva de unión observada represente una titulación de unión de alta afinidad siMás que una verdadera isoterma vinculante. Si esto se observa, repetir el ensayo utilizando una concentración de actina F 10 veces menor para medir una afinidad precisa. Para las interacciones de alta afinidad, la estabilización de la faloidina (etapa 3.3) puede ser necesaria para lograr una concentración de actina F lo suficientemente baja como para medir con precisión la afinidad. Finalmente, existen limitaciones fundamentales con el ensayo de co-sedimentación que los investigadores deben tener en cuenta al realizar y evaluar el ensayo. Más importante aún, el ensayo de co-sedimentación no produce una constante de equilibrio real. Los productos de unión ( es decir, proteína más F-actina) se separan de los reactivos durante la centrifugación, después de lo cual los productos pueden disociarse para crear un nuevo equilibrio. Como resultado, el ensayo de co-sedimentación puede calcular erróneamente o no detectar interacciones de baja afinidad. Puesto que muchas proteínas de unión a actina tienen una afinidad baja ( es decir, micromolar) para la actina F, un resultado negativo <eM&gt; es decir, ninguna unión detectable) en el ensayo no significa necesariamente que una proteína no se une a F-actina. Como alternativa, los ensayos de unión a un solo filamento basados ​​en microscopía TIRF son más sensibles y más precisos para determinar una constante de disociación (para revisiones sobre esta técnica, véanse las referencias 15 , 16 ). A pesar de estas limitaciones, el ensayo de granulación está dentro de los medios de la mayoría de los investigadores y es una herramienta eficaz para determinar si una proteína se une a la actina F y para medir la afinidad de la interacción.

La actina es una proteína esencial del citoesqueleto que desempeña un papel crítico en múltiples procesos celulares, incluyendo la motilidad, la contracción, la adhesión y la morfología 1 . La actina existe en dos formas: la actina globular monomérica (G-actina) y la actina filamentosa polimerizada (F-actina). Dentro de las células, F-actina organización está controlada por una gran colección de proteínas que regulan la nucleación, el crecimiento, reticulación y desmontaje de actina filamentos 2 , 3 , 4 . Sin embargo, la forma en múltiples actina vinculante proteínas funcionan en concierto para regular la actina red de organización sigue siendo en gran parte poco clara. La medición de las interacciones proteína-proteína es un enfoque importante para entender cómo las proteínas ejercen sus efectos sobre el comportamiento celular a nivel bioquímico. Se pueden usar muchos ensayos diferentes para detectar interacciones entre proteínas purificadas.Los enfoques comunes para las proteínas solubles incluyen pull-downs, polarización de fluorescencia, calorimetría de titulación isotérmica y resonancia de plasmón superficial. Es importante destacar que todos estos ensayos requieren que las proteínas sean solubles y, por lo tanto, son difíciles de adaptar para su uso con una proteína polimérica filamentosa tal como actina F. Aquí, describimos una técnica - la co-sedimentación de actina, o granulación, ensayo - para determinar si una proteína o dominio de proteínas se une a la actina F y para medir la afinidad de la interacción. El ensayo de granulación de actina es una técnica relativamente sencilla que no requiere equipo especializado, aparte de una ultracentrífuga. Todos los reactivos se pueden hacer, asumiendo el conocimiento de la bioquímica básica, o comprado. Una vez establecida la unión a F-actina, el ensayo puede usarse para medir la afinidad aparente ( es decir, la constante de equilibrio de disociación) 5 . Además, una vez que se establece una afinidad, el ensayo de granulaciónEs una herramienta útil para medir cómo los cambios en la proteína de interés ( es decir , las modificaciones post-traduccionales, mutaciones, ligando o ligando) afectan a su interacción con F-actina [ 6] . La técnica tiene limitaciones (vea la Discusión ) que el investigador debe tener en cuenta antes de intentar el ensayo.

<table><tbody><tr><td>Sorvall MTX 150 Micro-Ultracentrifuge</td><td>ThermoFisher Scientific</td><td>46960</td><td></td></tr><tr><td>S100-AT3 rotor</td><td>ThermoFisher Scientific</td><td>45585</td><td></td></tr><tr><td>Ultracentrifuge tubes - 0.2 mL</td><td>ThermoFisher Scientific</td><td>45233</td><td></td></tr><tr><td>Actin, rabbit skeletal muscle</td><td>Cytoskeleton</td><td>AKL99</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A8531</td><td></td></tr><tr><td>Polidicanol (Thesit)&amp;nbsp;</td><td>Sigma</td><td>88315</td><td></td></tr><tr><td>Phalloidin</td><td>ThermoFisher Scientific</td><td>P3457</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>ThermoFisher Scientific</td><td>R0862</td><td></td></tr><tr><td>Adenosine triphosphate (ATP)</td><td>Sigma</td><td>A2383</td><td></td></tr><tr><td>Imidazole</td><td>Fisher Scientific</td><td>O3196</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>M33</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217</td><td></td></tr><tr><td>Ethylene glycol-bis(&amp;beta;-aminoethyl ether)-N,N,N&#039;,N&#039;-tetraacetic acid (EGTA)</td><td>Sigma</td><td>3779</td><td></td></tr><tr><td>Odyssey CLx Imaging System</td><td>LI-COR</td><td></td><td></td></tr><tr><td>Coomassie Brilliant Blue R-250 Dye</td><td>ThermoFisher Scientific</td><td>20278</td><td></td></tr><tr><td>Colloidal Blue Staining Kit</td><td>ThermoFisher Scientific</td><td>LC6025</td><td></td></tr></tbody></table>

measuring protein binding to f-actin by co-sedimentation

La organización de actina filamentosa (F-actina) dentro de las células está regulada por un gran número de proteínas de unión a actina que controlan la nucleación, crecimiento, reticulación y / o desmontaje de actina. Este protocolo describe una técnica - la co-sedimentación de actina, o granulación, ensayo - para determinar si un dominio de proteína o proteína se une a F-actina y para medir la afinidad de la interacción ( es decir, la constante de equilibrio de disociación). En esta técnica, una proteína de interés se incuba primero con F-actina en solución. A continuación, se utiliza la centrifugación diferencial para sedimentar los filamentos de actina, y el material en forma de gránulo se analiza por SDS-PAGE. Si la proteína de interés se une a la actina F, se co-sedimentará con los filamentos de actina. Los productos de la reacción de unión ( es decir, F-actina y la proteína de interés) se pueden cuantificar para determinar la afinidad de la interacción. El ensayo de granulación de actina es una técnica sencilla para determinarSi una proteína de interés se une a la actina F y para evaluar cómo los cambios en esa proteína, como la unión al ligando, afectan su interacción con la F-actina.

Se examinó αE-catenina homodímero vinculante a F-actina en el co-sedimentación ensayo. Dado que los experimentos anteriores han demostrado que la afinidad del homodímero αE-catenina para la actina F es alrededor de 1 μM y el Bmax cerca de 11 , se realizó el ensayo con una concentración baja de actina F (0,2 μM en lugar de 2 μM; La discusión). Puesto que 0,2 μM está por debajo de la concentración crítica, se añadió faloidina para estabilizar la F-actina polimerizada a partir de G-actina de músculo esquelético de conejo (etapa 3.3). Se incubaron concentraciones crecientes de homodímero αE-catenina (0,125-12,0 μM) en presencia o ausencia de actina F 0,2 μM. Las muestras fueron centrifugadas, y los pellets resultantes se analizaron ( Figura 1A ]. Como era de esperar, el homodímero αE-catenina co-sedimentado con F-actina por encima del fondo ( Figura 1A , compara las muestras de pellet de F-actina con el no-F-acMuestras de pellets de hojalata). BSA se ejecutó como un control negativo ( Figura 1B ]. La proteína unida se cuantificó y se trazó sobre proteína libre para calcular la afinidad de la interacción ( Figura 1C ). Los datos trazados se ajustan mejor a una ecuación Hill. El Kd calculado fue de 2,9 mu M, el Bmax fue de 0,2 y el coeficiente de Hill (h) fue de 4,8. Por tanto, el homodímero αE-catenina se une a la actina F cooperativamente con una afinidad micromolar baja, consistente con el trabajo previo (un Kd de 2,9 μM frente a 1,0 μM) 11 . <img alt="Figura 1" src="/files/ftp_upload/55613/55613fig1.jpg" /> Figura 1: Ensayo de co-sedimentación de F-actina de alta velocidad. ( A ) Se incubaron concentraciones crecientes (0,125-12,0 mu M) de homodímero de alpha - catenina con (paneles de la izquierda) o sin (paneles de la derecha) 0,2 mu M de actina F estabilizada con phalloidinorte. Se incubaron durante 30 min a TA y se centrifugaron. El total (7,5% del material de partida) y el material granulado (50% del material en forma de gránulo) se separaron por SDS-PAGE y se tiñeron con colorante Coomassie. ( B ) 4 μ M BSA se ejecutó como un control negativo. Las muestras totales y de pellets, con (+) o sin (-) F-actina, se separaron por SDS-PAGE y se tiñeron con colorante de Coomassie. ( C ) La α-catenina unida (μM / μM de actina) de A se representó contra la α-catenina libre (μM) y los datos se ajustaron a una ecuación de Hill (línea roja). Se enumeran los coeficientes Kd, Bmax y Hill (h). <a href="http://ecsource.jove.com/files/ftp_upload/55613/55613fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/55613/55613fig2.jpg" /> Figura 2: Cuantificación de la peletización con actina </sTrong&gt; - diagrama de flujo. Este esquema describe los pasos clave en la sección 5, con ejemplos de muestras de Total y Pellet ( A , CE ) y la curva estándar ( B ) utilizada para la cuantificación. 5.13 pasos: 1 ) Medir la cantidad de proteína de interés en las muestras totales (A). 2 ) Genere una curva estándar trazando la intensidad de banda frente a la masa de proteína (B). 3 ) Medir la cantidad de proteína de interés que co-sedimentado con F-actina ( C ). 4 ) Mida la cantidad de proteína de interés que sedimentó en ausencia de F-actina ( D ). 5 ) Sustraiga D de C para determinar la cantidad de proteína unida a F-actina. 6 ) Mida la cantidad de F-actina en cada pellet (E), calcule la cantidad promedio de F-actina por muestra y divida cada muestra por el promedio (los números abajo muestran la relación). 7 )Para cada muestra, divida la cantidad de proteína unida (calculada en el paso 5) por la proporción de gránulos de actina F (calculada en el paso 6) para ajustar las diferencias en el gránulo. 8 ) Utilice la curva estándar ( B ) para calcular la cantidad (masa) de proteína unida normalizada en cada muestra (paso 7). 9 ) Determine la concentración de proteína libre y proteína unida para crear una curva de unión. <a href="http://ecsource.jove.com/files/ftp_upload/55613/55613fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Measuring Protein Binding to F-actin by Co-sedimentation at JoVE.com

Measuring Protein Binding to F-actin by Co-sedimentation

Este protocolo describe un método para probar la capacidad de una proteína para co-sedimentar con actina filamentosa (F-actina) y, si se observa unión, para medir la afinidad de la interacción.

Medición de la unión de proteínas a actina F por co-sedimentación

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

measuring-protein-binding-to-f-actin-by-co-sedimentation

Research

JoVE Journal

Biochemistry

16.4K vistas.  University of Pittsburgh School of Medicine.  Este protocolo describe un método para probar la capacidad de una proteína para co-sedimentar con actina filamentosa (F-actina) y, si se observa unión, para medir la afinidad de la interacción. 

Video: Measuring Protein Binding to F-actin by Co-sedimentation

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

La anisotropía de fluorescencia como una herramienta para estudiar las interacciones proteína-proteína

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

Investigación

Bioquímica

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo- and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single &#945;-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein. While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured.&#160;&#160;

Sedimentation equilibrium (SE) can be used to study protein-protein interactions in a physiological environment. This manuscript describes the use of this technique to determine the effect of pH on the stability of a homo-pentamer formed by the small hydrophobic (SH) protein encoded by the human syncytial respiratory virus (hRSV).

La sedimentación de equilibrio de una proteína de membrana pequeña formador de oligómeros: Efecto de la histidina protonación en pentamérica Estabilidad

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

Química

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Biología

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Un análisis de espectroscopia de fluorescencia fluctuación de interacciones de proteínas en los contactos célula-célula

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

Desde un 2DE-Gel Spot para Función de Proteínas: Lección aprendida de HS1 en la leucemia linfocítica crónica

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

Medicina

Rapid Assessment of Membrane Protein Quality by Fluorescent Size Exclusion Chromatography

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different tags, truncations, deletions, fusion partner insertions, and stabilizing mutations to find one that is not aggregated after extraction from the membrane. Furthermore, buffer screening to determine the detergent, additive, ligand, or polymer that provides the most stabilizing condition for the membrane protein is an important practice. The early characterization of membrane protein quality by fluorescent size exclusion chromatography provides a powerful tool to assess and rank different constructs or conditions without the requirement for protein purification, and this tool also minimizes the sample requirement. The membrane proteins must be fluorescently tagged, commonly by expressing them with a GFP tag or similar. The protein can be solubilized directly from whole cells and then crudely clarified by centrifugation; subsequently, the protein is passed down a size exclusion column, and a fluorescent trace is collected. Here, a method for running FSEC and representative FSEC data on the GPCR targets sphingosine-1-phosphate receptor (S1PR1) and serotonin receptor (5HT2AR) are presented.

The present protocol describes a procedure to perform fluorescent size exclusion chromatography (FSEC) on membrane proteins to assess their quality for downstream functional and structural analysis. Representative FSEC results collected for several G-protein coupled receptors (GPCRs) under detergent-solubilized and detergent-free conditions are presented.

Evaluación rápida de la calidad de la proteína de membrana mediante cromatografía de exclusión de tamaño fluorescente

During membrane protein structural elucidation and biophysical characterization, it is common to trial numerous protein constructs containing different ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Ensayo de sedimentación basado en concanavalina A para medir la unión al sustrato de las fosfatasas de glucano

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Reconstitución de la motilidad basada en actina con proteínas disponibles comercialmente

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

Bioingeniería

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.

Polymerization of FtsZ is essential for bacterial cell division. In this report, we detail simple protocols to monitor FtsZ polymerization activity and discuss the influence of buffer composition. The protocols can be used to study the interaction of FtsZ with regulatory proteins or antibacterial drugs that affect FtsZ polymerization.

FtsZ Polimerización Ensayos: Protocolos simples y Consideraciones

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Cuantificación de interacciones de proteínas basada en imágenes de molécula única de células vivas

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

Autor destacado: Evaluación de la dinámica proteína-condensado en células humanas vivas

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

Medición de una sola molécula de la dinámica de interacción de proteínas dentro de condensados biomoleculares

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...