Este trabalho foi financiado em partes pelo NIH concede GM073197 e RR025336.

Um esquema típico de um experimento em meso cristalização é mostrado na Fig.1 1,2. Pré-cristalização LCP FRAP-ensaios são opcionais, no entanto, eles podem acelerar significativamente o processo de busca de condições de cristalização inicial, especialmente no caso de proteínas da membrana difícil 3. 1. Reconstituição de proteínas em LCP <ol><li> Purificar uma proteína de membrana de interesse em uma solução de detergente e concentrar a complexos de proteína / detergente para ~ 10 - 20 mg / mL, tomando cuidado para não sobre-concentrar a 1,4 detergente. </li><li> Transferência de ~ 25 mg de um lipídio anfitrião LCP (tipicamente monoolein) ou uma mistura de lipídios em um tubo de 1,5 mL de plástico e incubar a 40 ° C por alguns minutos até que os lipídios se derrete. </li><li> Anexar um acoplador de seringa para uma seringa de 100 mL à prova de gás. </li><li> Carregar a seringa com a lipídios fundido usando uma pipeta de volume ajustável. Registrar o volume dos lipídios na seringa. </li><li> Carregar outro seringa de 100 L com a solução de proteína em uma proteína relação de solução em lipídeos 03/02 v / v. </li><li> Conectar ambas as seringas em conjunto, através do acoplador seringa. </li><li> Empurre a seringa êmbolos alternadamente para mover a lipídios e proteínas, através da agulha interior do engate, e para trás, até que a mesofase lipídios torna-se homogênea. LCP formas espontaneamente ao mistura mecânica, ea proteína torna-se reconstituído na bicamada lipídica da LCP. Formação de LCP pode ser verificada por sua consistência transparente e gel-like e pela ausência de birrefringência quando visto sob um microscópio equipado com cross-polarizadores, ou, se possível, usando pequeno ângulo de difração de raios-X 1. </li></ol> 2. LCP-FRAP pré-cristalização Ensaios LCP-FRAP testes são projetados para medir as propriedades de difusão de proteínas da membrana reconstituída em LCP em uma variedade de condições de triagem 3. A difusão de longo alcance de proteínas da membrana em LCP é essencial para a cristalização de sucesso, no entanto, a microestrutura do LCP restringe a difusão de proteínas ou grandes agregados da proteína oligomérica. Uma razão comum para o fracasso de um experimento em meso cristalização é uma agregação de proteínas rápida levando a uma perda de difusão. Tem sido demonstrado que o comportamento de agregação de uma proteína depende da construção de determinada proteína, lipídios do hospedeiro e da composição da solução de triagem 3. <ol><li> Rótulo a proteína com um corante fluorescente (Cy3 ou similar) na proporção de proteína / tintura de ~ 100 / 1, remover o corante não reagido e concentrar a proteína ~ 1 mg / mL. Rótulo ou aminas livres ou tióis livres. Quando rotulagem aminas livres, use pH entre 7 e 7,5 a etiqueta predominantemente livre N-terminal. Esteja ciente de que a rotulagem amino também pode rótulo lipídios co-purificada com a proteína 2,3. </li><li> Reconstituir a proteína rotulados de LCP, conforme descrito na seção 1). </li><li> Configurar placas de ensaio como descrito na seção 3) usando soluções LCP-FRAP triagem em vez de telas de cristalização 2. </li><li> Incubar as placas a 20 ° C no escuro por pelo menos 12 horas para alcançar um estado de equilíbrio. </li><li> Coloque uma das placas na estação LCP-FRAP e foco no primeiro poço usando uma objetiva de 10x. </li><li> Aquisição de 5 imagens fluorescentes para capturar o estado pré-branqueada inicial. </li><li> Acionar o laser. A potência do laser e do número de pulsos deve ser ajustada para bleach ~ 30 - 70% da proteína rotulado no meio da mancha branqueada. </li><li> Imediatamente após o disparo do laser, iniciar a gravação de uma seqüência de pós-clareamento rápido de ~ 200 imagens a uma taxa mais rápida possível. </li><li> Siga com a gravação de uma seqüência de pós-bleach-lenta de ~ 50 imagens, selecionando a demora entre imagens como s 20/01, dependendo da taxa de difusão da proteína. </li><li> Integrar a intensidade dentro do local de lixívia em todos os quadros e corrigi-lo para o branqueamento ea luz flutuações de intensidade durante a aquisição, dividindo a intensidade dentro do local branqueada pela intensidade média de um ponto de referência fora da área de laser branqueada. </li><li> Normalizar a intensidade corrigida para tornar a intensidade pré-branqueada igual a 1 e a intensidade inicial branqueada igual a 0. </li><li> O traçado da curva da intensidade normalizada em função do tempo, F (t), usando a seguinte equação 5: F (t) = M x exp (-2T / t) x (I 0 (2T / t) + I 1 (2T / t)), (Eq.1) onde M é a fração de moléculas de difusão móvel, T é o tempo de difusão característico, t é o tempo real de cada quadro gravado, I 0 e I 1 são os dias 0 e 1 ª ordem modificado funções de Bessel. </li><li> Calcular o coeficiente de difusão, D, como: D = R 2 / 4T, (Eq.2) onde R é o raio da mancha branqueada. </li><li> Mover to bem próxima e repita os passos 2.5) - 2.13). </li><li> Compare as frações móvel e coeficientes de difusão obtidos para as condições de seleção diferentes. Concepção nova cristalização telas com base nos componentes que facilitaram a difusão de proteínas e as condições de exclusão para o qual a difusão de proteína não foi observada. Se a proteína não difusa em qualquer uma das condições selecionadas, considerar ampliando o espaço de triagem ou tentando construir uma nova proteína. </li></ol> 3. Configurando Trials Cristalização LCP <ol><li> Proteína em reconstituir a LCP, conforme descrito na seção 1). </li><li> Transferir a LCP proteína-laden em uma seringa de 10 mL à prova de gás ligado a uma seringa dispenser repetitivo. </li><li> Anexar uma pequena agulha removível (calibre 26, 10 mm de comprimento) para a seringa de 10 mL. </li><li> Dispense 200 nL bolo de LCP na superfície de quatro poços adjacentes formando um quadrado 2x2. </li><li> Sobreposição de cada um dos bolos LCP com 1 mL de solução correspondente a cristalização tela. </li><li> Cap quatro poços carregado com uma lamela de vidro 18 milímetros quadrados. Aplique uma pressão suave sobre as lamelas para selar os poços. </li><li> Repita os passos 3,4) -3,6) com o próximo conjunto de 4 poços até a placa inteira está cheia. </li><li> Incubar a placa a uma temperatura constante, verificando periodicamente a formação de cristais e crescimento. </li></ol> 4. Cristais colheita de LCP <ol><li> Coloque um prato com cristais de proteínas sob um microscópio estéreo com zoom variável, equipado com um polarizador e analisador linear de rotação. </li><li> Concentre-se no bem de interesse usando um zoom de baixa potência para que o bem geral é colocado dentro do campo de visão. </li><li> Pontuação do vidro lamela em quatro cursos fazendo um quadrado dentro dos limites bem usando um canto de uma pedra afiada corte de cerâmica capilar. </li><li> Imprensa em torno do perímetro marcou com fortes sharp-ponto pinças para propagar os riscos através da espessura do vidro lamela. </li><li> Faça dois pequenos furos em cantos opostos do quadrado marcado. </li><li> Injetar mL de solução precipitante poucos através de um dos buracos para reduzir a desidratação durante as etapas subseqüentes. </li><li> Usando uma sonda de agulha em ângulo agudo quebrar o vidro ao longo de um ou dois lados para liberar a praça cut-out. </li><li> Cuidadosamente levante a assistir quadrados de vidro para o bolo fase cúbicos. Se o bolo está preso à lamela, em seguida, virar o quadrado de vidro e coloque no fundo do poço. </li><li> Adicionar um extra alguns mL de solução precipitante, completada com uma crio-protetor, se necessário, no topo do bolo fase expostos cúbicos no poço. </li><li> Aumentar a ampliação do microscópio e se concentrar em um cristal. </li><li> Ajustar o ângulo entre o polarizador eo analisador para aumentar o contraste entre o cristal birrefringente eo fundo, mantendo luz suficiente para ver o circuito de colheita. </li><li> Selecione um MicroMount MiTeGen com um diâmetro correspondente ao tamanho de cristal e depois colher o cristal diretamente da LCP escavando-o na MicroMount. </li><li> Flash congelar o MicroMount com o cristal colhidas em nitrogênio líquido, e enviá-lo a uma linha de luz síncrotron fonte para coleta de dados de raios-X 6. </li></ol> 5. Resultados representativos: Uma engenharia humana beta 2 adrenérgicos G receptor acoplado à proteína (β 2 AR-T4L) foi expressa em baculovírus infectados células de inseto Sf9 e purificado em dodecylmaltoside (DDM) / hemissuccinato colesterol (CHS) solução de detergente vinculado a um agonista parcial inversa carazololo 7. A proteína foi marcado com Cy3 éster NHS e utilizado em ensaios pré-cristalização LCP-FRAP (Figura 2). Telas grade grosseira com base em várias condições selecionados a partir dos resultados da LCP-FRAP ensaios produzidos inicial cristal-como hits (Figura 3). Otimização das condições de precipitante rendeu cristais de difração de qualidade (Figura 4). <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="Figura 1" /> Figura 1. Fluxograma de um experimento típico LCP cristalização. Passos nas caixas cinza não estão descritos nos protocolos atuais. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="Figura 2" /> Figura 2. LCP-ensaio FRAP com AR-T4L/carazolol 2 β em monoolein LCP baseado. A) Resultados de um ensaio FRAP LCP-realizada em um modo de alto rendimento automático, em que cada amostra de uma placa de 96 poços é branqueada seqüencialmente e recuperação de fluorescência é medida após a incubação de 30 min. As recuperações obtidas por fluorescência, que representam a fração móvel em cada amostra, são traçados para todas as 96 amostras. As soluções de triagem contêm 0,1 M Tris pH 8, 30% v / v PEG 400 combinado com 48 diferentes sais em duas concentrações diferentes. B) Fluorescência perfis de recuperação para vários repcondições representativas. Curvas linha sólida representa encaixa pela equação. 1.O frações móvel e os coeficientes de difusão são determinados utilizando equações. 1 e 2. Recuperação rápida de menos de 10% na amostra contendo cloreto de sódio é devido a lipídios fluorescente etiquetado co-purificada com a proteína. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="Figura 3" /> Figura 3. Sucessos cristal inicial de AR-T4L/carazolol 2 β obtido por uma triagem grade grossa em torno de condições mais promissoras identificadas pela LCP-FRAP, contendo sulfato de sódio (painel A) e Na Formate (painel B). A proteína é rotulado com Cy3 éster NHS e as imagens fluorescentes são tiradas com excitação em 543 nm e emissão a 605 nm. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="Figura 4" /> Figura 4. Cristais Optimized de AR-T4L/carazolol 2 β. As imagens dos cristais crescidos na presença de sulfato de sódio (painéis A e B) e K Formate (painéis C e D) são tomadas no modo brightfield (painéis A e C) e usando cross-polarizadores (painéis 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. 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>

Não há conflitos de interesse declarados.

Os protocolos de fornecer uma orientação básica visual para as principais etapas envolvidas na realização de experimentos de cristalização meso. Mais em profundidade os detalhes relacionados a estes protocolos, enfatizando possíveis armadilhas, deficiências ou rotas alternativas estão disponíveis em outros lugares 1,2. Opcional LCP FRAP-ensaios podem ajudar nas fases anteriores para selecionar a construção de proteínas mais promissores, LCP lipídica host e aditivos de lipídios, bem como limitar a gama de possíveis precipitantes e condições de buffer 3. Uma vez um sucesso de cristalização inicial é encontrada, ela deve ser otimizado para obter cristais de melhor qualidade. Otimização das condições de cristalização de meso é essencialmente similar a otimização das condições de proteínas solúveis com a adição de parâmetros adicionais associados com a composição da LCP 1. Cristais de proteína de membrana lipídica cultivadas em mesofase são geralmente menores em tamanho do que os cristais obtidos em solução de detergente, mas mais ordenada, assim, beneficiando fortemente do uso de linhas de luz disponíveis em microfoco fontes de síncrotron moderna 6. Muitos dos procedimentos relacionados no meso cristalização, incluindo a configuração de placas de cristalização ou ensaio, a realização de LCP FRAP-ensaios e detecção de cristais, foram semi ou totalmente automatizado 1,2,8,9, permitindo triagem de uma grande variedade de condições ao consumir pequenas quantidades de proteínas e lipídios. Por outro lado, proteína em reconstituições LCP e colheita de cristal permanecem operações manuais e mais tedioso e, portanto, têm uma necessidade de melhoria.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

Proteínas da membrana realizar funções críticas em células vivas relacionadas com o transporte do sinal de transdução, e transformações de energia, e, como tal, estão implicados em uma infinidade de disfunções e doenças. No entanto, um entendimento estrutural e funcional de proteínas da membrana é fortemente ficando muito atrás de seus parceiros solúveis, principalmente, devido às dificuldades associadas à sua solubilização e geração de cristais de qualidade de difração. Cristalização em mesofases lipídico (também conhecido como cristalização em meso ou LCP) é uma técnica promissora que foi aplicado com sucesso a obtenção de estruturas de alta resolução de rodopsinas microbiana, proteínas fotossintéticas, barris beta membrana externa e acoplados à proteína G receptores. Em meso cristalização tira proveito de um ambiente de membrana nativa-like e, normalmente, produz cristais com menor teor de solvente e melhor organização, em comparação com a cristalização das soluções tradicionais de detergente. O método não é difícil, mas requer uma compreensão do comportamento de lipídios fase e prática na manipulação de materiais viscosos mesofase. Aqui nós demonstramos uma forma simples e eficiente de fazer LCP e reconstituindo uma proteína de membrana na bicamada lipídica da LCP usando um mixer seringa, seguida de distribuição de porções nanolitros de LCP em um ensaio ou placa de cristalização, a realização de ensaios de pré-cristalização e colheita de cristais a matriz LCP. Esses protocolos fornecem um guia básico para a abordagem em ensaios de cristalização meso, no entanto, como em qualquer experimento de cristalização, uma extensa triagem e otimização são necessárias, e um resultado bem-sucedido não é necessariamente garantido.

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

Crystallization of Membrane Proteins in Lipidic Mesophases

Os protocolos descrevem os passos essenciais para a obtenção de cristais de difração de qualidade de uma proteína de membrana a partir de reconstituição da proteína em uma fase lipídica cúbicos (LCP), encontrando condições iniciais com os ensaios pré-cristalização LCP-FRAP, a criação de ensaios de cristalização LCP e cristais colheita .

Cristalização de proteínas da membrana lipídica em mesofases

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

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

Research

JoVE Journal

Biology

31.2K visualizações.  The Scripps Research Institute. Os protocolos descrevem os passos essenciais para a obtenção de cristais de difração de qualidade de uma proteína de membrana a partir de reconstituição da proteína em uma fase lipídica cúbicos (LCP), encontrando condições iniciais com os ensaios pré-cristalização LCP-FRAP, a criação de ensaios de cristalização LCP e cristais colheita .

Vídeo: 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.

Cristalizando Proteínas de Membrana para determinação da estrutura usando mesofases lipídico

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

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.

Avaliar Trials bidimensional cristalização de proteínas da membrana Pequenas de Estudos Biologia Estrutural por Cristalografia 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.

Examinando o Dynamics Conformacional de Proteínas de Membrana In situ Com Labeling-Site dirigido Fluorescência

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

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

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

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

De alto rendimento cristalização de proteínas de membrana Usando o método Bicelle lipídico

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.

A utilização de um robô para alta capacidade de cristalização de proteínas de membrana em mesofases lipídicas

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

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

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

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

Colheita e Cryo-resfriamento cristais de proteínas de membrana crescida no mesofases lipídicas para a determinação da estrutura de Cristalografia Macromolecular

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

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

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

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

Microscopia intravital para Estruturas de imagem Subcelulares em ratos vivos Expressando fluorescentes Proteínas

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.

Imunodetecção de membrana externa proteínas por citometria de fluxo de mitocôndrias isoladas

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.

Verde Fluorescente à base de proteínas Expressão Rastreio de Proteínas de Membrana em<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.

Microperf Técnica para Investigar Regulamento de microvasos Permeabilidade de Rat Mesentery

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