Ce travail a été financé en partie par les subventions des NIH et de GM073197 RR025336.

Un plan typique d&#39;une expérience de cristallisation dans le méso est montré à la Fig.1 1,2. Pré-cristallisation LCP-FRAP tests sont facultatifs, mais ils peuvent considérablement accélérer le processus de recherche de conditions de cristallisation initiale, particulièrement dans le cas des protéines membranaires difficiles 3. 1. Reconstitution des protéines dans les LCP <ol><li> Purifier une protéine membranaire d&#39;intérêt dans une solution détergente et de concentrer les complexes protéine / détergent pour ~ 10 - 20 mg / ml, en prenant soin de ne pas trop concentrer les 1,4 détergent. </li><li> Transfert ~ 25 mg d&#39;un lipide d&#39;hôte LCP (généralement monooléine) ou un mélange de lipides dans un tube de 1,5 ml en plastique et incuber à 40 ° C pendant quelques minutes jusqu&#39;à ce que les lipides fond. </li><li> Fixez un coupleur de la seringue pour une 100 uL étanche seringue. </li><li> Charger la seringue avec le lipide fondu en utilisant une pipette de volume réglable. Noter le volume du lipide dans la seringue. </li><li> Charger une autre seringue 100 pL avec la solution de protéine à une protéine solution à des lipides rapport 2 / 3 v / v. </li><li> Connectez les deux seringues ensemble à travers le coupleur seringue. </li><li> Poussez la seringue pistons en alternance pour déplacer les lipides et de protéines grâce à l&#39;aiguille interne du coupleur, en arrière, jusqu&#39;à ce que la mésophase lipides devient homogène. LCP forme spontanément lors du mélange mécanique, et la protéine devient reconstituée dans la bicouche lipidique du PAFR. Formation du LCP peut être vérifiée par sa cohérence et de transparence gel, et par l&#39;absence de biréfringence lorsqu&#39;on les examine sous un microscope équipé d&#39;polariseurs croisés, ou, si possible, en utilisant X aux petits angles de diffraction des rayons 1. </li></ol> 2. LCP-FRAP pré-cristallisation Tests LCP-FRAP tests sont conçus pour mesurer les propriétés de diffusion des protéines membranaires reconstitués dans LCP à une variété de conditions de dépistage 3. La diffusion à longue distance de protéines membranaires dans LCP est essentiel pour la cristallisation de succès, cependant, la microstructure de la LCP contraintes de diffusion des protéines de grande taille ou d&#39;agrégats de protéines oligomériques. Une raison fréquente de l&#39;échec d&#39;une expérience de cristallisation dans le méso est une agrégation de protéines rapide conduisant à une perte de diffusion. Il a été démontré que le comportement d&#39;agrégation d&#39;une protéine dépend de la construction d&#39;une protéine particulière, le lipide hôte et la composition de la solution 3 de dépistage. <ol><li> Étiquette de la protéine avec un colorant fluorescent (Cy3 ou similaire) à un ratio protéines / colorant de ~ 100 / 1, éliminer le colorant qui n&#39;a pas réagi et se concentrer à la protéine de ~ 1 mg / mL. Étiquette soit amines libres ou thiols libres. Lors de l&#39;étiquetage des amines libres, utilisez pH entre 7 et 7,5 à prédominance label gratuite N-terminale. Soyez conscient que l&#39;étiquetage aminés peuvent également l&#39;étiquette lipides co-purifiée avec la protéine 2,3. </li><li> Reconstituer la protéine étiquetée LCP comme décrit dans la section 1). </li><li> Mettre en place des plaques de dosage comme décrit dans la section 3) en utilisant des solutions de dépistage LCP-FRAP au lieu de 2 écrans cristallisation. </li><li> Incuber les plaques à 20 ° C dans l&#39;obscurité pendant au moins 12 heures pour atteindre un état d&#39;équilibre. </li><li> Placez l&#39;une des plaques sur la station de LCP-FRAP et se concentrer sur le premier puits en utilisant un objectif 10x. </li><li> Acquérir 5 images fluorescentes pour capturer les premières pré-blanchi de l&#39;Etat. </li><li> Déclencher le laser. La puissance du laser et du nombre d&#39;impulsions doit être ajustée à l&#39;eau de Javel ~ 30 - 70% de la protéine marquée au milieu de la tache blanchie. </li><li> Immédiatement après le déclenchement du laser, commencer à enregistrer un rapide post-séquence de blanchiment de ~ 200 images à la cadence la plus rapide possible. </li><li> Suivez avec l&#39;enregistrement d&#39;une lente après-javel séquence de ~ 50 images, la sélection du délai entre les images que 10 à 20 s, selon le taux de diffusion de la protéine. </li><li> Intégrer l&#39;intensité à l&#39;intérieur de l&#39;endroit l&#39;eau de Javel dans tous les cadres et les corriger pour le blanchiment et la lumière des fluctuations d&#39;intensité lors de l&#39;acquisition en divisant l&#39;intensité à l&#39;intérieur de la tache blanchie par l&#39;intensité moyenne d&#39;un spot de référence en dehors de la zone au laser blanchi. </li><li> Normaliser l&#39;intensité corrigée pour rendre l&#39;intensité de pré-blanchi égal à 1 et l&#39;intensité initiale blanchi égal à 0. </li><li> Monter la courbe de l&#39;intensité normalisée en fonction du temps, F (t), en utilisant l&#39;équation suivante 5: F (t) = M x exp (-2T / t) x (I 0 (2T / t) + I 1 (2T / t)), (Eq.1) où M est la fraction mobile de molécules diffusantes, T est le temps caractéristique de diffusion, t est le temps réel de chaque image enregistrée, I 0 et I 1 sont les e 0 et 1 er afin fonctions de Bessel modifiées. </li><li> Calculer le coefficient de diffusion, D, comme: D = R 2 / 4T, (Eq.2) où R est le rayon de la tache blanchie. </li><li> Déplacez to le bien suivant et répétez les étapes 2,5) - 2,13). </li><li> Comparez les fractions mobiles et les coefficients de diffusion obtenus pour les conditions de dépistage différents. Conception nouvelle cristallisation des écrans basés sur les composants qui ont facilité la diffusion des protéines et des conditions d&#39;exclusion pour lesquelles la diffusion de protéines n&#39;a pas été observé. Si la protéine ne diffusent pas dans l&#39;une des conditions dépistage, envisager d&#39;élargir l&#39;espace de dépistage ou d&#39;essayer de construire une nouvelle protéine. </li></ol> 3. Configuration des essais de cristallisation LCP <ol><li> Reconstituer la protéine dans LCP comme décrit dans la section 1). </li><li> Transfert du LCP protéines chargées dans un 10 uL étanche seringue fixée à un distributeur de seringues répétitives. </li><li> Fixer une aiguille courte amovible (calibre 26, longueur 10 mm) à la seringue de 10 mL. </li><li> Distribuer 200 nL bolus de LCP à la surface de quatre puits adjacents formant un carré de 2x2. </li><li> Superposition de chacun des bolus LCP avec 1 ul de solution de cristallisation écran correspondant. </li><li> Cap quatre puits chargé avec une lamelle de verre de 18 mm carrés. Appliquez une légère pression sur la lamelle pour sceller les puits. </li><li> Répétez les étapes 3.4) -3.6) avec la prochaine série de 4 puits jusqu&#39;à ce que la plaque entière est remplie. </li><li> Incuber la plaque à une température constante, et vérifie périodiquement la formation de cristaux et de croissance. </li></ol> 4. Récolte Cristaux de LCP <ol><li> Placer une plaque avec des cristaux de protéines sous un microscope stéréo avec zoom variable, équipé d&#39;un polariseur linéaire tournante et analyseur. </li><li> Focus sur le bien d&#39;intérêt en utilisant un zoom de faible puissance afin que le bien tout est placé dans le champ de vision. </li><li> Score le verre lamelle en quatre coups faisant un carré à l&#39;intérieur des limites bien en utilisant un angle vif d&#39;une pierre de coupe capillaire en céramique. </li><li> Appuyer sur le pourtour marqué avec une forte forte-point des pincettes pour propager les rayures grâce à l&#39;épaisseur du verre couvre-objet. </li><li> Percez deux petits trous aux coins opposés du carré marqué. </li><li> Injecter uL quelques solution de précipitant à travers un des trous pour réduire la déshydratation au cours des étapes ultérieures. </li><li> En utilisant une sonde d&#39;aiguille à angle pointu briser le verre le long d&#39;un ou deux côtés pour libérer la place découpe. </li><li> Soulevez délicatement le regarder carrés de verre pour le bolus phase cubique. Si le bolus est collé à la lamelle, puis retournez le carré de verre et placez sur le fond du puits. </li><li> Ajouter un supplément de quelques uL d&#39;une solution de précipitant, complété par un cryo-protecteur, si nécessaire, au-dessus du bol phase cubique exposés dans le puits. </li><li> Augmenter l&#39;agrandissement du microscope et de se concentrer sur un cristal. </li><li> Ajustez l&#39;angle entre le polariseur et l&#39;analyseur pour augmenter le contraste entre le cristal biréfringent et l&#39;arrière-plan, tout en gardant assez de lumière pour voir la boucle de récolte. </li><li> Sélectionner un MicroMount MiTeGen avec un diamètre correspondant à la taille des cristaux et ensuite récolter le cristal directement à partir du LCP en l&#39;excavation dans le MicroMount. </li><li> Flash geler les MicroMount avec le cristal récoltés dans l&#39;azote liquide, et l&#39;expédier à une ligne de lumière synchrotron à rayons X de collecte de données 6. </li></ol> 5. Les résultats représentatifs: Une conçu beta 2-adrénergiques humains G récepteurs couplés aux protéines (β 2 AR-T4L) a été exprimée dans le baculovirus cellules d&#39;insectes Sf9 infectées et purifié dans dodecylmaltoside (DDM) / cholestérol hémisuccinate (SHC) une solution de détergent lié à un agoniste partiel inverse carazolol 7. La protéine a été marquée avec Cy3 ester NHS et utilisés dans les LCP-FRAP pré-cristallisation des dosages (Figure 2). Écrans grille grossière basée sur plusieurs conditions sélectionnées à partir des résultats des tests de LCP-FRAP produite initiaux semblables à des cristaux frappe (figure 3). Optimisation des conditions de précipitation a donné des cristaux par diffraction de qualité (figure 4). <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="Figure 1" /> Figure 1. Organigramme d&#39;une expérience de cristallisation LCP typique. Étapes dans les cases grises ne sont pas décrites dans les protocoles actuels. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="Figure 2" /> Figure 2. LCP-FRAP dosage avec β 2 AR-T4L/carazolol dans monooléine LCP base. A) les résultats d&#39;un essai LCP-FRAP effectuées à une machine automatique à haut débit en mode, dans lequel chaque échantillon d&#39;une plaque de 96 puits est blanchie séquentiellement et de recouvrement de fluorescence est mesurée après une incubation de 30 min. Le taux de récupération obtenus par fluorescence, qui représentent la fraction mobile dans chaque échantillon, sont tracées pour tous les 96 échantillons. Les solutions de criblage contiennent Tris 0,1 M pH 8, 30% v / v PEG 400 combiné avec 48 différents sels à deux concentrations différentes. B) Les profils de recouvrement de fluorescence pour rep plusieurssentant des conditions. Courbes en trait plein représentent s&#39;adapte par l&#39;Eq. 1.L fractions mobiles et les coefficients de diffusion sont déterminés en utilisant les équations. 1 et 2. La récupération rapide de moins de 10% dans l&#39;échantillon contenant du chlorure de Na est due aux lipides marqués par fluorescence co-purifiée avec la protéine. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="Figure 3" /> Figure 3. Coups de cristal initiale de β 2 AR-T4L/carazolol obtenue par une grille grossière de dépistage autour de la plupart des conditions prometteuses identifiées par LCP-FRAP, contenant du sulfate de Na (panneau A) et Na Formiate (panneau B). La protéine est étiqueté avec Cy3 ester NHS et les images fluorescentes sont prises en utilisant une excitation à 543 nm et émission à 605 nm. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="Figure 4" /> Figure 4. Cristaux optimisée des β 2 AR-T4L/carazolol. Les images de cristaux cultivés en présence de sulfate de Na (panneaux A et B) et K formate (panneaux C et D) sont prises en mode fond clair (panneaux A et C) et en utilisant polariseurs croisés (panneaux B et 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>

Les protocoles de fournir une orientation de base visuel pour les principales étapes à effectuer dans les expériences de cristallisation méso. Plus en profondeur les détails relatifs à ces protocoles, en soulignant les pièges possibles, des lacunes ou des itinéraires alternatifs sont disponibles nulle part ailleurs 1,2. En option LCP-FRAP tests peuvent aider lors des phases antérieures de choisir le concept le plus prometteur de protéines, de lipides et d&#39;additifs d&#39;hôte LCP lipides, ainsi que limiter la gamme des facteurs déclenchants possibles et les conditions de tampon 3. Une fois un succès cristallisation initiale est trouvé, il devrait être optimisé pour obtenir des cristaux de meilleure qualité. Optimisation des conditions de cristallisation dans le méso est essentiellement similaire à l&#39;optimisation des conditions pour les protéines solubles avec l&#39;ajout de paramètres supplémentaires associés à la composition de LCP 1. Des cristaux de protéines membranaires cultivées en mésophase lipidique sont généralement de plus petite taille que les cristaux obtenus dans une solution détergente, mais plus ordonnée, par conséquent, bénéficier fortement de l&#39;aide microfoyer faisceaux disponibles auprès de sources synchrotron moderne 6. La plupart des procédures liées à la cristallisation méso, y compris la mise en place des plaques de cristallisation ou de dosage, la conduite LCP-FRAP tests et la détection des cristaux, ont été semi ou entièrement automatique 1,2,8,9, permettant le dépistage d&#39;une large gamme de conditions tout en consommant de petites quantités de protéines et de lipides. D&#39;autre part, les reconstitutions de protéines dans LCP et la récolte de cristal reste des opérations manuelles fastidieuses et plus et, par conséquent, ont un besoin d&#39;amélioration.

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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>
		</div>

crystallization of membrane proteins in lipidic mesophases

Les protéines membranaires exécuter des fonctions critiques dans les cellules vivantes liées à la transduction du signal, le transport et les transformations d&#39;énergie, et, comme tels, sont impliqués dans une multitude de dysfonctionnements et maladies. Cependant, une compréhension structurale et fonctionnelle des protéines membranaires est fortement à la traîne de leurs partenaires solubles, principalement en raison de difficultés liées à leur solubilisation et la génération de cristaux de qualité de diffraction. Cristallisation dans mésophases lipidique (également connu sous le nom de la cristallisation méso ou LCP) est une technique prometteuse qui a été appliquée avec succès à obtenir des structures à haute résolution de rhodopsins microbienne, les protéines photosynthétiques, extérieur barils beta membrane et G récepteurs couplés aux protéines. En méso cristallisation profite d&#39;un environnement de membrane native-like et produit généralement avec des cristaux de la teneur en solvant inférieure et une meilleure commande par rapport à la cristallisation traditionnels de solutions détergentes. La méthode n&#39;est pas difficile, mais nécessite une compréhension du comportement phase lipidique et la pratique dans la manutention des matériaux mésophase visqueux. Ici nous démontrons une manière simple et efficace de faire de LCP et la reconstitution d&#39;une protéine membranaire dans la bicouche lipidique du LCP utilisant un mélangeur à seringue, suivie par la distribution des portions du nanolitre LCP dans un essai ou une plaque de cristallisation, la conduite des essais de pré-cristallisation et la récolte des cristaux à partir la matrice LCP. Ces protocoles fournissent un guide de base pour aborder dans les essais de cristallisation méso; cependant, comme avec n&#39;importe quel expérience de cristallisation, le dépistage étendu et l&#39;optimisation sont nécessaires, et le succès n&#39;est pas nécessairement garantie.

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

Crystallization of Membrane Proteins in Lipidic Mesophases

Les protocoles décrivent les étapes essentielles pour obtenir des cristaux par diffraction de la qualité d&#39;une protéine membranaire à partir de la reconstitution de la protéine dans une phase lipidique cubique (LCP), trouver les conditions initiales avec LCP-FRAP pré-cristallisation des essais, la mise en place des essais de cristallisation LCP et des cristaux de récolte .

La cristallisation des protéines membranaires dans des mésophases lipidique

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

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31.2K vues.  The Scripps Research Institute. Les protocoles décrivent les étapes essentielles pour obtenir des cristaux par diffraction de la qualité d'une protéine membranaire à partir de la reconstitution de la protéine dans une phase lipidique cubique (LCP), trouver les conditions initiales avec LCP-FRAP pré-cristallisation des essais, la mise en place des essais de cristallisation LCP et des cristaux de récolte .

Vidéo: 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.

Cristallisant protéines membranaires pour déterminer la structure à l&#39;aide mésophases lipidique

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

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Biologie

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.

L&#39;évaluation des essais de cristallisation bidimensionnelle de protéines membranaires petites pour les études en biologie structurale par cristallographie électronique

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.

L&#39;examen de la dynamique conformationnelle des protéines membranaires In situ Avec site-dirigé marquage fluorescent

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.

Haut débit cristallisation des protéines membranaires en utilisant la méthode lipidique 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.

Utilisation d&#39;un robot pour haut-débit cristallisation des protéines membranaires dans des mésophases lipidiques

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.

Cristaux de récolte et de Cryo-refroidissement de protéines membranaires cultivées dans des mésophases lipidiques pour la détermination de la structure par cristallographie macromoléculaire

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.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines 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.

Immunodétection de la membrane externe de protéines par cytométrie en flux de mitochondries isolées

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.

Green Fluorescent Protein base Expression dépistage des protéines membranaires dans<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.

Microperfusion Technique chargé d&#39;enquêter sur le règlement des microvaisseaux perméabilité au Rat Mesentery

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