Name: crystallization of membrane proteins in lipidic mesophases
Uploaded: 2011-03-28T12:00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Questo lavoro è stato finanziato in parte dal NIH GM073197 e RR025336.

Un profilo tipico di un esperimento di cristallizzazione meso è mostrato in Fig.1 1,2. Pre-cristallizzazione LCP-FRAP saggi sono opzionali, tuttavia, possono notevolmente accelerare il processo di ricerca di condizioni di cristallizzazione iniziali, soprattutto nel caso delle proteine ​​di membrana difficile 3. 1. La ricostituzione delle proteine ​​in LCP <ol><li> Purificare una proteina di membrana di interesse in una soluzione detergente e concentrare la proteina / detergente complessi di ~ 10 - 20 mg / mL, facendo attenzione a non concentrare troppo il 1,4 detergente. </li><li> Trasferimento ~ 25 mg di lipidi ospitare LCP (tipicamente monoolein) o una miscela dei lipidi in un tubo di 1,5 ml di plastica e incubare a 40 ° C per qualche minuto fino a quando la lipidica si scioglie. </li><li> Collegare un accoppiatore siringa ad un 100 microlitri a tenuta di gas siringa. </li><li> Caricare la siringa con la lipidico fuso con una pipetta del volume regolabile. Registrare il volume del lipidi nella siringa. </li><li> Carico di altri 100 microlitri siringa con la soluzione proteica in un rapporto proteina soluzione-to-lipidico 2 / 3 v / v. </li><li> Collegare i due siringhe insieme attraverso l&#39;accoppiatore siringa. </li><li> Spingere la siringa tuffatori alternativamente per spostare il lipidi e proteine ​​attraverso l&#39;ago interna del giunto, avanti e indietro, fino a quando la mesofase lipidi diventa omogenea. LCP si forma spontaneamente sulla miscelazione meccanica, e la proteina viene ricostituita nel doppio strato lipidico del LCP. Formazione di LCP può essere verificata dalla sua coerenza trasparente e gelatinosa e per l&#39;assenza di birefringency se visto sotto un microscopio dotato di cross-polarizzatori, o, se possibile, utilizzando piccole angolo diffrazione di raggi X 1. </li></ol> 2. LCP-FRAP pre-cristallizzazione saggi LCP-FRAP test sono progettati per misurare le proprietà di diffusione delle proteine ​​di membrana ricostituite in LCP a una varietà di condizioni di screening 3. Il lungo raggio diffusione delle proteine ​​di membrana in LCP è essenziale per la cristallizzazione di successo, tuttavia, la microstruttura di LCP vincola la diffusione delle proteine ​​di grandi dimensioni o aggregati di proteine ​​oligomeriche. Un motivo comune per il fallimento di un esperimento di cristallizzazione meso è una aggregazione proteica veloce che porta ad una perdita di diffusione. E &#39;stato dimostrato che il comportamento di aggregazione di una proteina dipende dalla costruzione particolare proteina, i lipidi di accoglienza e la composizione della soluzione di screening 3. <ol><li> Etichetta la proteina con un colorante fluorescente (Cy3 o simili) con un rapporto proteine ​​/ colorante di circa 100 / 1, rimuovere il colorante che non ha reagito e concentrare la proteina a ~ 1 mg / mL. Etichetta sia ammine libero o tioli liberi. Quando l&#39;etichettatura ammine libero, l&#39;uso pH tra 7 e 7,5 per etichettare prevalentemente la libera N-terminale. Essere consapevoli del fatto che l&#39;etichettatura aminoacidi possono anche etichetta lipidi co-purificato con la proteina 2,3. </li><li> Ricostituire la proteina etichettato in LCP, come descritto nella sezione 1). </li><li> Istituito piatti test come descritto nella sezione 3) utilizzando LCP-FRAP soluzioni di screening, invece di schermi cristallizzazione 2. </li><li> Incubare le piastre a 20 ° C al buio per almeno 12 ore per raggiungere uno stato di equilibrio. </li><li> Mettere una delle piastre sul LCP-FRAP stazione e concentrarsi sul primo pozzo utilizzando un obiettivo 10x. </li><li> Acquisire 5 immagini a fluorescenza per catturare l&#39;iniziale stato di pre-sbiancato. </li><li> Innescare il laser. La potenza del laser e il numero di impulsi deve essere regolato a candeggina ~ 30 - 70% delle proteine ​​etichettate nel mezzo della macchia sbiancato. </li><li> Immediatamente dopo l&#39;attivazione del laser, iniziare a registrare un rapido post-candeggio sequenza di circa 200 immagini al tasso più veloce possibile. </li><li> Seguite con la registrazione di un lento post-candeggina sequenza di ~ 50 immagini, scegliendo il ritardo tra le immagini di 1-20 s, a seconda della velocità di diffusione della proteina. </li><li> Integrare l&#39;intensità all&#39;interno della macchia di candeggina in tutti i fotogrammi e correggere per il candeggio e fluttuazioni di intensità della luce durante l&#39;acquisizione dividendo l&#39;intensità all&#39;interno della macchia sbiancato con l&#39;intensità media di un punto di riferimento al di fuori della zona di laser sbiancato. </li><li> Normalizzare l&#39;intensità corretta per effettuare la pre-sbiancato intensità uguale a 1 e l&#39;intensità iniziale sbiancato uguale a 0. </li><li> Montare la curva della intensità normalizzata in funzione del tempo, F (t), utilizzando la seguente equazione 5: F (t) = M x exp (-2T / t) x (I 0 (2T / t) + I 1 (2T / t)), (Eq.1) dove M è la frazione mobile di molecole che diffondono, T è il tempo di diffusione caratteristica, t è il tempo reale di ogni fotogramma registrato, I 0 e I 1 sono lo 0 ° e 1 ° ordine modificate funzioni di Bessel. </li><li> Calcolare il coefficiente di diffusione, D, come: D = R 2 / 4T, (Eq.2) dove R è il raggio dello spot sbiancato. </li><li> Spostare to il pozzo successivo e ripetere i punti 2,5) - 2.13). </li><li> Confrontare le frazioni cellulari e coefficienti di diffusione ottenuti per le diverse condizioni di screening. Cristallizzazione nuovo design schermi in base ai componenti che hanno facilitato la diffusione delle proteine ​​e le condizioni di esclusione per i quali la diffusione delle proteine ​​non è stata osservata. Se la proteina non ha diffuso in alcuna delle condizioni di screening, cercare di estendere lo spazio di screening o cercare una nuova proteina costrutto. </li></ol> 3. Impostazione Prove di cristallizzazione LCP <ol><li> Ricostituire la proteina in LCP, come descritto nella sezione 1). </li><li> Trasferire il carico di proteine ​​LCP in un 10 microlitri a tenuta di gas siringa collegata ad un erogatore siringa ripetitivo. </li><li> Inserire un ago corto rimovibile (calibro 26, 10 mm di lunghezza) alla siringa 10 L. </li><li> Dispensare 200 boli nL del LCP sulla superficie di quattro pozzi adiacenti che formano un quadrato 2x2. </li><li> Sovrapposizione ciascuno dei boli LCP con 1 ml di soluzione corrispondente schermo cristallizzazione. </li><li> Cap quattro pozzi caricato con un coprioggetto 18 mm di vetro quadrati. Applicare una leggera pressione sul vetrino per sigillare i pozzi. </li><li> Ripetere i passaggi 3.4) -3,6), con la prossima serie di 4 pozzi fino a quando il piatto è pieno. </li><li> Incubare la piastra a temperatura costante, controllando periodicamente per la formazione di cristalli e di crescita. </li></ol> 4. La raccolta Cristalli da LCP <ol><li> Posizionare un piatto con cristalli proteici al microscopio stereo con zoom variabile, dotato di un polarizzatore lineare di rotazione e analizzatore. </li><li> Concentrarsi sul pozzo di interesse utilizzando uno zoom a bassa potenza in modo che il tutto è ben posizionato all&#39;interno del campo visivo. </li><li> Punteggio ottenuto il vetro coprioggetto in quattro colpi facendo un quadrato all&#39;interno dei confini bene con un angolo acuto di una pietra da taglio in ceramica capillare. </li><li> Stampa in tutto il perimetro segnato con una forte forte punto di pinzette per propagare i graffi attraverso lo spessore del vetro coprioggetto. </li><li> Punch due piccoli fori agli angoli opposti della piazza segnato. </li><li> Iniettare pochi microlitri di soluzione precipitante attraverso uno dei fori per ridurre la disidratazione durante le fasi successive. </li><li> Utilizzando una sonda angolata ago appuntito rompere il vetro lungo uno o due lati per liberare il cut-out quadrati. </li><li> Sollevare delicatamente il quadrato di vetro per guardare il bolo fase cubi. Se il bolo è bloccato al coprioggetti, poi capovolgere il quadrato di vetro sopra e posto sul fondo del pozzo. </li><li> Aggiungi un ulteriore pochi microlitri di soluzione precipitante, integrato con un crio-protettore, se necessario, sulla parte superiore del bolo esposti fase cubi nel pozzo. </li><li> Aumentare l&#39;ingrandimento del microscopio e concentrarsi su un cristallo. </li><li> Regolare l&#39;angolo tra il polarizzatore e l&#39;analizzatore per aumentare il contrasto tra il cristallo birifrangente e lo sfondo, mantenendo abbastanza luce per vedere il ciclo di raccolta. </li><li> Selezionare un micromount MiTeGen con un diametro corrispondente al formato di cristallo e poi raccogliere il cristallo direttamente dal LCP scavando in la micromount. </li><li> Flash congelare il micromount con i cristalli raccolti in azoto liquido, e la nave ad una linea di luce di sincrotrone per la fonte di raggi X raccolta di dati 6. </li></ol> 5. Rappresentante dei risultati: Un progettato umano beta 2 adrenergici G protein-coupled receptor (β 2 AR-T4L) è stato espresso in baculovirus infette SF9 cellule di insetto e purificata in dodecylmaltoside (DDM) / colesterolo emisuccinato (CHS) una soluzione detergente associato a un agonista parziale inversa carazololo 7. La proteina è stato marcato con Cy3 estere NHS e utilizzato in LCP-FRAP pre-cristallizzazione test (Figura 2). Schermi griglia grossolana sulla base di diverse condizioni selezionate dai risultati di LCP-FRAP saggi prodotto iniziale di cristallo come colpi (Figura 3). Ulteriore ottimizzazione delle condizioni precipitanti ceduto cristalli di qualità diffrazione (Figura 4). <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="Figura 1" /> Figura 1. Flow-chart di un tipico esperimento di cristallizzazione LCP. Passi nel box grigio non sono descritte nella attuali protocolli. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="Figura 2" /> Figura 2. LCP-FRAP test con β 2 AR-T4L/carazolol in LCP monoolein base. A) i risultati di un LCP-FRAP test eseguiti in automatico la modalità high-throughput, in cui ogni campione di una piastra a 96 pozzetti è sbiancato in sequenza e il recupero fluorescenza viene misurata dopo una incubazione di 30 min. I recuperi ottenuti fluorescenza, che rappresentano la frazione mobile in ogni campione, sono tracciati per tutti i 96 campioni. Le soluzioni di screening contengono 0,1 M Tris pH 8, 30% v / v PEG 400 in combinazione con 48 diversi sali a due differenti concentrazioni. B) profili di fluorescenza di recupero per alcuni repcondizioni rappresentativi. Curve linea continua rappresenta adatta da Eq. 1.L frazioni mobile e coefficienti di diffusione sono determinati utilizzando equazioni. 1 e 2. Recupero veloce di meno del 10% del campione contenente cloruro di sodio è dovuto al lipidi fluorescente co-purificato con la proteina. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="Figura 3" /> Figura 3. Colpi di cristallo iniziale di β 2 AR-T4L/carazolol ottenuto dalla proiezione griglia grossolana attorno condizioni più promettenti identificati da LCP-FRAP, contenenti Na solfato (pannello A) e Na formiato (pannello B). La proteina è marcato con Cy3 estere NHS e le immagini fluorescenti sono scattate con eccitazione a 543 nm ed emissione a 605 nm. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="Figura 4" /> Figura 4. Cristalli ottimizzata di β 2 AR-T4L/carazolol. Le immagini dei cristalli cresciuti in presenza di solfato di Na (pannelli A e B) e K formiato (pannelli C e D) sono presi in modalità campo chiaro (pannelli A e C) e l&#39;utilizzo di polarizzatori incrociati (pannelli B e 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. 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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. 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Nessun conflitto di interessi dichiarati.

I protocolli di fornire una guida di base visivo per le tappe principali coinvolti nello svolgimento di esperimenti di cristallizzazione meso. Più approfondito i dettagli relativi a questi protocolli, sottolineando possibili insidie, carenze o percorsi alternativi sono disponibili altrove 1,2. Opzionale LCP-FRAP test può aiutare nelle fasi precedenti per selezionare il costrutto proteina più promettenti, lipidi ospitare LCP e additivi lipidico, così come limitare la gamma di possibili fattori precipitanti e le condizioni di buffer 3. Una volta che un colpo di cristallizzazione iniziale si trova, dovrebbe essere ottimizzato per ottenere cristalli di migliore qualità. Ottimizzazione delle condizioni di cristallizzazione in meso è essenzialmente simile a ottimizzare le condizioni per le proteine ​​solubili con l&#39;aggiunta di parametri aggiuntivi associati con la composizione del LCP 1. Cristalli di proteina di membrana coltivate in mesofase lipidico sono generalmente di dimensioni inferiori rispetto cristalli ottenuti in soluzione detergente, ma più ordinato, quindi, beneficiando fortemente usare i microfocus linee di luce disponibile al moderne sorgenti di sincrotrone 6. Molte delle procedure relative alla cristallizzazione in meso, compresa la creazione di piatti cristallizzazione o saggio, conducendo LCP-FRAP test e rilevazione cristalli, sono state parzialmente o completamente automatizzati 1,2,8,9, che consente lo screening di una vasta gamma di condizioni consumando piccole quantità di proteine ​​e lipidi. D&#39;altra parte, ricostruzioni proteina in LCP e la raccolta di cristallo rimangono le operazioni manuali e più noioso e, quindi, hanno bisogno di miglioramento.

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		<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

Proteine ​​di membrana svolgono funzioni fondamentali nelle cellule viventi relativi alla trasduzione del segnale, il trasporto e trasformazioni di energia, e, come tali, sono coinvolti in una moltitudine di disfunzioni e malattie. Tuttavia, una comprensione strutturale e funzionale delle proteine ​​di membrana è fortemente arretrato rispetto a quello dei loro partner solubile, soprattutto, a causa delle difficoltà legate alla loro solubilizzazione e la generazione di cristalli di qualità diffrazione. Cristallizzazione in mesophases lipidico (noto anche come cristallizzazione meso o LCP) è una tecnica promettente che è stato applicato con successo per ottenere strutture ad alta risoluzione di rhodopsins microbica, proteine ​​fotosintetiche, botti beta della membrana esterna e G recettori accoppiati alla proteina. In meso cristallizzazione si avvale di un nativo ambiente simile a membrana e produce tipicamente cristalli con basso contenuto di solventi e meglio ordinare rispetto ai tradizionali cristallizzazione da soluzioni detergenti. Il metodo non è difficile, ma richiede una comprensione del comportamento dei lipidi fase e pratiche nel trattamento dei materiali mesofase viscosi. Qui mostriamo un modo semplice ed efficiente di fare LCP e la ricostituzione di una proteina di membrana nel doppio strato lipidico di LCP utilizzando un mixer siringa, seguita dalla distribuzione di porzioni nanoliter LCP in un saggio o un piatto di cristallizzazione, conducendo i test pre-cristallizzazione e la raccolta di cristalli l&#39;LCP matrice. Questi protocolli di fornire una guida di base per accostarsi a prove di cristallizzazione meso, tuttavia, come per qualsiasi esperimento di cristallizzazione, lo screening esteso e ottimizzazione sono necessarie, e un risultato positivo non è necessariamente garantita.

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

Crystallization of Membrane Proteins in Lipidic Mesophases

I protocolli di descrivere le fasi essenziali per ottenere cristalli di diffrazione qualità di una proteina di membrana a partire dalla ricostituzione della proteina in una fase lipidica cubi (LCP), trovando condizioni iniziali con LCP-FRAP pre-cristallizzazione analisi, la creazione di processi di cristallizzazione LCP e cristalli di raccolta .

Cristallizzazione delle proteine ​​di membrana lipidica in Mesophases

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

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.

Cristallizzare proteine ​​di membrana per la determinazione della struttura utilizzando Mesophases lipidica

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

Ricerca

Biologia

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.

Valutazione bidimensionale Prove di cristallizzazione delle proteine ​​di membrana per piccoli studi di biologia strutturale di Cristallografia Electron

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.

Esaminando la dinamica conformazionale delle proteine ​​di membrana In situ Con il sito-diretta Etichettatura fluorescenza

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.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica 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.

L&#39;utilizzo di un robot per High-throughput cristallizzazione delle proteine ​​di membrana in mesofasi lipidici

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.

Cristalli di raccolta e di Cryo-raffreddamento delle proteine ​​di membrana Grown in mesofasi lipidici per la determinazione della struttura di cristallografia macromolecolare

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.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

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.

Immunolocalizzazione della membrana esterna proteine ​​mediante citometria di flusso di mitocondri isolati

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 Espressione Proiezione di proteine ​​di membrana in<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.

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

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

Cristallizzazione delle proteine ​​di membrana lipidica in Mesophases

Cristallizzazione delle proteine di membrana lipidica in Mesophases