Este trabajo fue financiado en parte por las subvenciones del NIH GM073197 y RR025336.

Un esquema típico de un experimento de cristalización meso se muestra en la Fig. 1 1,2. Cristalización pre-FRAP LCP-ensayos son opcionales, sin embargo, que puede acelerar significativamente el proceso de búsqueda de condiciones de cristalización inicial, especialmente en el caso de las proteínas de la membrana difícil 3. 1. La reconstitución de proteínas en la LCP <ol><li> Purificar una proteína de membrana de interés en una solución de detergente y se concentran los complejos de proteínas / detergente a ~ 10 - 20 mg / ml, teniendo cuidado de no sobre-concentrar el 1,4 detergente. </li><li> Transferencia de ~ 25 mg de lípidos LCP anfitrión (por lo general monoolein) o una mezcla de lípidos en un tubo de plástico de 1,5 ml y se incuba a 40 ° C durante unos minutos hasta que los lípidos se derrita. </li><li> Conecte un acoplador de la jeringa para un 100 L hermético jeringa. </li><li> Cargue la jeringa con el lípido fundido utilizando una pipeta de volumen variable. Registrar el volumen de los lípidos en la jeringa. </li><li> Carga otra jeringa 100 L con la solución de proteína en una solución de proteínas a los lípidos proporción 2 / 3 v / v. </li><li> Conecte los dos jeringas juntos a través del acoplador de la jeringa. </li><li> Empuje el émbolo alternativamente jeringa para mover los lípidos y proteínas a través del interior de la aguja del acoplador, de ida y vuelta, hasta que la mesofase lípidos se vuelve homogéneo. LCP se forma espontáneamente en la mezcla mecánica, y la proteína se reconstituye en la bicapa lipídica de la LCP. Formación de LCP puede ser verificado por su consistencia gelatinosa y transparente, y por la ausencia de birrefringencia cuando se observan bajo un microscopio equipado con polarizadores cruzados, o, si es posible, mediante el uso de un pequeño ángulo de difracción de rayos X-1. </li></ol> 2. LCP-FRAP cristalización pre-ensayos LCP-FRAP ensayos están diseñados para medir las propiedades de difusión de las proteínas de la membrana se reconstituye en LCP en una variedad de condiciones de selección 3. La difusión de largo alcance de las proteínas de membrana de LCP es esencial para la cristalización de éxito, sin embargo, la microestructura de la LCP limita la difusión de proteínas de gran tamaño o de agregados de proteínas oligoméricas. Una razón común para el fracaso de un experimento de cristalización meso es una agregación de proteínas rápida que conduce a una pérdida de difusión. Se ha demostrado que el comportamiento de agregación de una proteína depende de la construcción de proteína en particular, los lípidos de acogida y la composición de la solución de detección 3. <ol><li> Etiqueta de la proteína con un tinte fluorescente (Cy3 o similar) en una proporción de proteína / tinte de ~ 100 / 1, quitar el tinte sin reaccionar y concentrar la proteína de ~ 1 mg / ml. Etiqueta, ya sea libre o aminas tioles libres. Cuando el etiquetado aminas libres, use pH entre 7 y 7,5 para la etiqueta predominantemente libres N-terminal. Tenga en cuenta que el etiquetado de amino también pueden etiquetar los lípidos co-purifica con la proteína de 2,3. </li><li> La reconstitución de la proteína marcada en LCP como se describe en la sección 1). </li><li> Configurar placas de ensayo como se describe en la sección 3) utilizando LCP-FRAP soluciones de control en lugar de dos pantallas de la cristalización. </li><li> Las placas se incuban a 20 ° C en la oscuridad durante al menos 12 horas para lograr un estado de equilibrio. </li><li> Coloque uno de los platos en la estación de LCP-FRAP y se centran en el primer pozo con un objetivo de 10x. </li><li> Adquirir 5 imágenes fluorescentes para capturar la primera pre-blanqueado estado. </li><li> Activar el láser. La potencia del láser y el número de impulsos se debe ajustar a la lejía ~ 30 - 70% de la proteína marcada en el centro de la mancha blanqueada. </li><li> Inmediatamente después de la activación del láser, iniciar la grabación de un ayuno posterior a la secuencia de blanqueo ~ 200 imágenes a la mayor velocidad posible. </li><li> Seguir con la grabación de una lenta después de la secuencia de blanqueo ~ 50 imágenes, seleccionar el tiempo entre las imágenes como 1.20 s, en función de la tasa de difusión de la proteína. </li><li> Integrar la intensidad en el interior del lugar de blanqueador en todos los marcos y corregirla para el blanqueo y la luz fluctuaciones de intensidad durante la adquisición de dividir la intensidad en el interior del lugar blanqueado por la intensidad promedio de un punto de referencia fuera del área de láser blanqueada. </li><li> Normalizar la intensidad corregida para que la intensidad de la pre-blanqueado igual a 1 y de la intensidad inicial blanqueada igual a 0. </li><li> Ajustar la curva de la intensidad normalizada en función del tiempo, F (t), utilizando la siguiente ecuación 5: F (t) = M x exp (-2T / t) x (I 0 (2T / t) + I 1 (2T / t)), (Eq.1) donde M es la fracción de moléculas que se difunden móvil, T es el tiempo de difusión característico, t es el tiempo real de cada fotograma grabado, I 0 y 1 son los 0 º y 1 º orden modificado las funciones de Bessel. </li><li> Calcular el coeficiente de difusión, D, como: D = R 2 / 4T, (ecuación 2) donde R es el radio de la mancha blanqueada. </li><li> No se muevao bien la siguiente y repita los pasos 2,5) - 2,13). </li><li> Compara las fracciones celulares y los coeficientes de difusión obtenidos para las condiciones de selección diferentes. Cristalización nuevo diseño basado en las pantallas de los componentes que facilitan la difusión de las proteínas y las condiciones de exclusión para que la difusión de las proteínas no se observó. Si la proteína no se difundieron en cualquiera de las condiciones de detección, estudiar la ampliación del espacio de proyección o está tratando de construir una nueva proteína. </li></ol> 3. Configuración de los ensayos de cristalización LCP <ol><li> La reconstitución de la proteína en la LCP como se describe en la sección 1). </li><li> La transferencia de la LCP proteína cargada en un 10 l impermeable a los gases jeringa conectada a un dispensador repetitivo jeringa. </li><li> Coloque una aguja corta extraíble (de calibre 26, 10 mm de longitud) a la jeringa l 10. </li><li> Prescindir de 200 bolos nL de LCP en la superficie de cuatro pozos adyacentes que forman un cuadrado de 2x2. </li><li> Superposición de cada uno de los bolos de LCP con una L de solución correspondiente pantalla de cristalización. </li><li> Cap cuatro pozos cargados con un cubreobjetos de 18 mm cuadrados. Aplique una presión suave sobre el cubreobjetos para sellar los pozos. </li><li> Repita los pasos 3,4) -3,6) con el siguiente grupo de 4 pozos hasta que el plato está lleno. </li><li> Incubar la placa a una temperatura constante, la comprobación periódica de la formación y crecimiento de cristales. </li></ol> 4. La cosecha de cristales LCP <ol><li> Coloque un plato con cristales de proteínas en un microscopio estéreo con zoom variable, equipado con un polarizador lineal de rotación y el analizador. </li><li> Centrarse en el bien de interés con un zoom de baja potencia para que el bien general se coloca en el campo de visión. </li><li> Puntuación del vidrio cubreobjetos en cuatro golpes haciendo un cuadrado dentro de los límites y con una esquina de una piedra de corte capilar de cerámica. </li><li> De prensa en todo el perímetro anotó con un fuerte fuerte unas pinzas de punta para propagar las rayas a través del espesor del vidrio cubreobjetos. </li><li> Perfore dos agujeros pequeños en las esquinas opuestas de la plaza, anotó. </li><li> L inyectar unos pocos de la solución de precipitación a través de uno de los agujeros para reducir la deshidratación durante las etapas posteriores. </li><li> Utilizando una sonda en ángulo aguja romper el vidrio a lo largo de uno o dos lados para liberar la plaza de desconexión. </li><li> Levante cuidadosamente la observación de cuadrados de vidrio para el bolo de fase cúbica. Si el bolo está atascada en el cubreobjetos, luego voltear el cuadrado de cristal y colóquelo en el fondo del pozo. </li><li> Añadir un extra l de la solución precipitante, complementado con un crio-protector, si es necesario, en la parte superior de la fase de bolo expuestos cúbicos en el pozo. </li><li> Aumentar la ampliación del microscopio y se centran en un cristal. </li><li> Ajustar el ángulo entre el polarizador y el analizador para aumentar el contraste entre el cristal birrefringente y el fondo, mientras se mantiene la luz suficiente para ver el circuito de la cosecha. </li><li> Seleccione un micromontajes MiTeGen con un diámetro que corresponde al tamaño de cristal y el cristal de la cosecha directamente de la LCP por el sacarla a la micromontajes. </li><li> Flash congelar la micromontajes con el cristal cosechado en nitrógeno líquido, y se lo enviaremos a una línea de luz sincrotrón para la fuente de rayos X de recogida de datos 6. </li></ol> 5. Los resultados representativos: Una ingeniería humana beta 2 adrenérgicos acoplados a proteínas G del receptor (β 2 AR-T4L) se expresó en baculovirus Sf9 infectadas con células de insectos y se purifica en dodecylmaltoside (DDM) / colesterol hemisuccinato (CHS) una solución de detergente unido a un agonista parcial inverso carazolol 7. La proteína fue marcado con Cy3 éster de NHS y utiliza LCP-FRAP cristalización pre-ensayos (Figura 2). Pantallas grueso de cuadrícula basado en varias condiciones seleccionadas de los resultados de la LCP-FRAP ensayos producidos inicial cristal-como golpes (Figura 3). Una mayor optimización de las condiciones de precipitación de cristales de difracción producido de la calidad (Figura 4). <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="Figura 1" /> Figura 1. Diagrama de flujo de un experimento de cristalización LCP típico. Medidas en los cuadros de color gris no están descritas en los protocolos actuales. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="Figura 2" /> Figura 2. LCP-FRAP ensayo con β 2 AR-T4L/carazolol en LCP monoolein base. A) Los resultados de un ensayo de LCP-FRAP realiza en un sistema automático de alto rendimiento el modo en el que cada muestra de una placa de 96 pocillos se blanquea de forma secuencial y la recuperación de la fluorescencia se mide después de una incubación de 30 minutos. Las recuperaciones obtenidas de fluorescencia, que representan la fracción de móviles en cada muestra, se trazan para todos 96 muestras. Las soluciones de detección contienen 0,1 M Tris pH 8, 30% v / v de PEG 400 en combinación con 48 diferentes sales en dos concentraciones diferentes. B) los perfiles de fluorescencia de recuperación de representante de variostante condiciones. Curvas sólidas representan la línea se ajusta a la ecuación. 1.El fracciones celulares y los coeficientes de difusión se determinan mediante las ecuaciones. 1 y 2. Rápida recuperación de menos del 10% de la muestra que contiene cloruro de sodio se debe a los lípidos marcados con fluorescencia co-purifica con la proteína. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="Figura 3" /> Figura 3. Éxitos iniciales de cristal de β 2 AR-T4L/carazolol obtenidos por un examen de la malla gruesa alrededor de las condiciones más prometedoras identificadas por LCP-FRAP, que contiene sulfato de sodio (panel A) y Na formiato (panel B). La proteína está marcado con Cy3 éster de NHS y las imágenes fluorescentes son tomadas con excitación a 543 nm y emisión a 605 nm. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="Figura 4" /> Figura 4. Optimizado cristales de β 2 AR-T4L/carazolol. Las imágenes de los cristales cultivados en presencia de sulfato de sodio (grupos A y B) y K-formato (paneles C y D) se toman en el modo de campo claro (grupos A y C) y el uso de polarizadores cruzados (los grupos B y D).

<ol>
	<li>Caffrey, M., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat. Protoc. 4, 706-731 (2009).</li><li>Cherezov, V., Abola, E., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-170 (2010).</li><li>Cherezov, V., Liu, J., Hanson, M. A., Griffith, M. T., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LCP-FRAP+assay+for+pre-screening+membrane+proteins+for+in+meso+crystallization.">LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization.</a> J. Cryst. Growth Design. 8, 4307-4315 (2008).</li><li>Misquitta, Y., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergents+destabilize+the+cubic+phase+of+monoolein:+implications+for+membrane+protein+crystallization.">Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.</a> Biophys. J. 79, 394-405 (2003).</li><li>Soumpasis, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+fluorescence+photobleaching+recovery+experiments.">Theoretical analysis of fluorescence photobleaching recovery experiments.</a> Biophys. J. 41, 95-97 (1983).</li><li>Cherezov, V., Hanson, M. A., Griffith, M. T., Hilgart, M. C., Sanishvili, R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rastering+strategy+for+screening+and+centering+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+micrometer+size+X-ray+synchrotron+beam.">Rastering strategy for screening and centering of microcrystal samples of human membrane proteins with a sub-10 micrometer size X-ray synchrotron beam.</a> J. R. Soc. Interface. 6, S587-S597 (2009).</li><li>Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robotic+system+for+crystallizing+membrane+and+soluble+proteins+in+lipidic+mesophases.">A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.</a> Acta Crystallogr. D. 60, 1795-1807 (2004).</li><li>Kissick, D. J., Gualtieri, E. J., Simpson, G. J., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+imaging+of+integral+membrane+protein+crystals+in+lipidic+mesophases.">Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.</a> Anal. Chem. 82, 491-497 (2010).</li><li>Cheng, A., Hummel, B., Qiu, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+mechanical+mixer+for+small+viscous+lipid-containing+samples.">A simple mechanical mixer for small viscous lipid-containing samples.</a> Chem. Phys. Lipids. 95, 11-21 (1998).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+inexpensive+nanoliter-volume+dispenser+for+highly+viscous+materials+used+in+membrane+protein+crystallization.">A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization.</a> J. Appl. Cryst. 38, 398-400 (2005).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</li><li>Cherezov, V., Fersi, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+screens:+compatibility+with+the+lipidic+cubic+phase+for+in+meso+crystallization+of+membrane+proteins.">Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.</a> Biophys J. 81, 225-242 (2001).</li></ol>

Los protocolos proporcionan una guía visual básica de los pasos principales involucrados en la realización de experimentos de cristalización meso. Más en profundidad los detalles relacionados con estos protocolos, con énfasis en las posibles dificultades, las deficiencias o las rutas alternativas están disponibles en otros lugares 1,2. Opcional FRAP LCP-ensayos pueden ayudar en las primeras etapas para seleccionar la construcción de las proteínas más prometedores, lípidos LCP de acogida y los aditivos de lípidos, así como limitar el rango de posibles factores desencadenantes y las condiciones de tampón 3. Una vez que un golpe de cristalización inicial se encuentra, debe ser optimizado para obtener una mejor calidad de los cristales. Optimización de las condiciones de cristalización meso es esencialmente similar a la optimización de las condiciones de las proteínas solubles con la adición de parámetros adicionales asociados con la composición de la LCP 1. Cristales de proteína de la membrana lipídica crecido en mesofase suelen ser de menor tamaño que los cristales obtenidos en solución de detergente, pero más ordenado, por lo tanto, se benefician fuertemente el uso de líneas de luz disponible en microfocus modernas fuentes de radiación sincrotrón 6. Muchos de los procedimientos relacionados con la cristalización meso, incluyendo la creación de placas de cristalización o ensayo, la realización de ensayos de LCP-FRAP y la detección de cristales, han sido semi-o completamente automático 1,2,8,9, lo que permite la proyección de un amplio rango de condiciones mientras que el consumo de pequeñas cantidades de proteínas y lípidos. Por otro lado, reconstituciones de proteínas en LCP y la cosecha de cristal permanecen las operaciones manuales y más tedioso y, por tanto, tienen una necesidad de mejora.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>100 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7656-01</td>
<td>2 syringes are required</td>
</tr>
<tr>
<td>10 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7653-01</td>
<td>&nbsp;</td>
<tr>
<td>Syringe coupler</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7770-02 30902</td>
<td>The coupler can be made using available parts as described in refs. 10</td>
</tr>
<tr>
<td>Repetitive syringe dispenser</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>83700</td>
<td>The repetitive syringe dispenser can be modified to reduce dispensing volume by ~3 times11</td>
</tr>
<tr>
<td>Short (0.375&#x201D;) flat-tipped removable needle (point style 3, gauge 26)</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7804-03</td>
<td>&nbsp;</td>
<tr>
<td>96-well glass sandwich plate</td>
<td>&nbsp;</td>
<td>Marienfeld</td>
<td>08 900 03</td>
<td>For manual operations it is more convenient to assemble the glass sandwich plate using a standard microscope glass slides and a double sticky tape with punched holes1,2,12.</td>
</tr>
<tr>
<td>Glass cover slip</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>63787-01</td>
<td>&nbsp;</td>
<tr>
<td>monoolein</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7765</td>
<td>&nbsp;</td>
<tr>
<td>Crystallization screens</td>
<td>&nbsp;</td>
<td>Hampton Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience</td>
<td>&nbsp;</td>
<td>Most of available commercial screens can be used for initial screening. Conditions that consistently disrupt LCP can be diluted 2x for better compatibility13.</td>
</tr>
<tr>
<td>Capillary cutting stone</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR4-334</td>
<td>&nbsp;</td>
<tr>
<td>Fine point tweezers</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>510</td>
<td>&nbsp;</td>
<tr>
<td>Angled sharp probe</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13650</td>
<td>&nbsp;</td>
<tr>
<td>MicroMounts</td>
<td>&nbsp;</td>
<td>MiTeGen</td>
<td>M1-Lxx-xx</td>
<td>Select MicroMount diameter to match the crystal size</td>
</tr>		</tbody>
		</table>
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crystallization of membrane proteins in lipidic mesophases

Las proteínas de membrana a cabo funciones críticas en las células vivas relacionadas con la transducción de señales, transporte y transformación de energía, y, como tal, están implicados en una multitud de disfunciones y enfermedades. Sin embargo, una comprensión estructural y funcional de proteínas de la membrana está muy a la zaga de sus socios soluble, principalmente, debido a dificultades relacionadas con su solubilización y la generación de los cristales de la calidad de difracción. La cristalización en mesofases lipídica (también conocida como la cristalización meso o LCP) es una técnica prometedora que se ha aplicado con éxito para obtener estructuras de alta resolución de rodopsinas microbianos, proteínas fotosintéticas, exterior barriles beta y los receptores de membrana acoplados a proteínas G. En meso cristalización se aprovecha de un ambiente de membrana como nativo y, normalmente, produce cristales con menor contenido en disolventes y ordenar mejor en comparación con los tradicionales de cristalización de soluciones detergentes. El método no es difícil, pero requiere una comprensión del comportamiento de los lípidos y la fase de prácticas en el manejo de materiales viscosos mesofase. Aquí se demuestra de una manera sencilla y eficaz de hacer LCP y la reconstitución de una proteína de membrana en la bicapa lipídica de la LCP utilizando un mezclador de jeringa, seguido por dispensar porciones nanolitros de LCP en un ensayo o una placa de cristalización, la realización de ensayos de pre-cristalización y la recolección de los cristales la matriz de LCP. Estos protocolos proporcionan una guía básica para abordar en los ensayos de cristalización meso, sin embargo, como con cualquier experimento de cristalización, la selección y optimización de extensas son necesarias, y el éxito no está garantizado necesariamente.

Watch this Scientific Journal Video about Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Crystallization of Membrane Proteins in Lipidic Mesophases

Los protocolos describen los pasos esenciales para la obtención de cristales de difracción de la calidad de una proteína de membrana a partir de la reconstitución de la proteína en una fase lipídica cúbicos (LCP), la búsqueda de las condiciones iniciales con LCP-FRAP antes de la cristalización de los ensayos, el establecimiento de ensayos LCP cristalización y los cristales de la cosecha .

La cristalización de proteínas de membrana en mesofases lipídico

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

crystallization-of-membrane-proteins-in-lipidic-mesophases

Research

JoVE Journal

Biology

31.2K vistas.  The Scripps Research Institute. Los protocolos describen los pasos esenciales para la obtención de cristales de difracción de la calidad de una proteína de membrana a partir de la reconstitución de la proteína en una fase lipídica cúbicos (LCP), la búsqueda de las condiciones iniciales con LCP-FRAP antes de la cristalización de los ensayos, el establecimiento de ensayos LCP cristalización y los cristales de la cosecha .

Video: Crystallization of Membrane Proteins in Lipidic Mesophases

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Cristalización de las proteínas de membrana para la Determinación de la estructura con mesofases lipídico

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Investigación

Biología

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

La evaluación de dos dimensiones ensayos de cristalización de proteínas de membrana pequeña de Estudios de Biología Estructural de Cristalografía de electrones

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examinar la dinámica conformacional de las proteínas de membrana In situ Sitio-dirigida con fluorescencia de etiquetado

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

El uso de un robot para la cristalización de alto rendimiento de proteínas de membrana en mesofases lipídicas

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Recolección y enfriamiento Cryo-cristales de proteínas de membrana cultivadas en mesofases lipídicas para la Determinación de la estructura por cristalografía macromolecular

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Microscopia intravital para formar imágenes de estructuras subcelulares en ratones vivos que expresan proteínas fluorescentes

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

La inmunodetección de la membrana externa proteínas por Citometría de Flujo de mitocondrias aisladas

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Fluorescente verde a base de proteína Expresión Proyección de proteínas de membrana en<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Microperfusión Técnica de investigar Reglamento de Microvessel Permeabilidad en Rata Mesenterio

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...