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	<li>Cole, K. S., Moore, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potassium+Ion+Current+in+the+Squid+Giant+Axon:+Dynamic+Characteristic.">Potassium Ion Current in the Squid Giant Axon: Dynamic Characteristic.</a> Biophys. J. 1, 1-14 (1960).</li><li>Taglialatela, M., Toro, L., Stefani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+voltage+clamp+to+record+small,+fast+currents+from+ion+channels+expressed+in+Xenopus+oocytes.">Novel voltage clamp to record small, fast currents from ion channels expressed in Xenopus oocytes.</a> Biophys. J. 61, 78-82 (1992).</li><li>Dempski, R. E., Friedrich, T., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+subunit+of+the+Na+/K+-ATPase+follows+the+conformational+state+of+the+holoenzyme.">The beta subunit of the Na+/K+-ATPase follows the conformational state of the holoenzyme.</a> J. Gen. Physiol. 125, 505-520 (2005).</li><li>Miller, A. J., Zhou, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+oocytes+as+an+expression+system+for+plant+transporters.">Xenopus oocytes as an expression system for plant transporters.</a> BBA - Biomembranes. 1465, 343-358 (2000).</li><li>Cha, A., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+Voltage-Dependent+Conformational+Changes+in+the+Shaker+K++Channel+with+Fluorescence.">Characterizing Voltage-Dependent Conformational Changes in the Shaker K+ Channel with Fluorescence.</a> Neuron. 19, 1127-1140 (1997).</li><li>Mannuzzu, L. M., Moronne, M. M., Isacoff, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Physical+Measure+of+Conformational+Rearrangement+Underlying+Potassium+Channel+Gating.">Direct Physical Measure of Conformational Rearrangement Underlying Potassium Channel Gating.</a> Science. 271, 213-216 (1996).</li><li>Koch, H. P., Larsson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-scale+molecular+motions+accomplish+glutamate+uptake+in+human+glutamate+transporters.">Small-scale molecular motions accomplish glutamate uptake in human glutamate transporters.</a> J. Neurosci. 25, 1730-1736 (2005).</li><li>Dempski, R. E., Hartung, K., Friedrich, T., Bamberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorometric+Measurements+of+Intermolecular+Distances+between+the+&#945;-+and+&#946;-Subunits+of+the+Na+/K+-ATPase.">Fluorometric Measurements of Intermolecular Distances between the &#945;- and &#946;-Subunits of the Na+/K+-ATPase.</a> J. Biol. Chem. 281, 36338-36338 (2006).</li><li>Geys, S. A., Bamberg, E., Dempski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-Dependent+Effects+on+the+Conformational+Equilibrium+of+the+Na+,K+-ATPase+As+Monitored+by+Voltage+Clamp+Fluorometry.">Ligand-Dependent Effects on the Conformational Equilibrium of the Na+,K+-ATPase As Monitored by Voltage Clamp Fluorometry.</a> Biophys. J. 96, 4561-4561 (2009).</li><li>Geibel, S., Kaplan, J. H., Bamberg, E., Friedrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+Dynamics+of+the+Na+/K+-ATPase+Probed+by+Voltage+Clamp+Fluorometry.">Conformational Dynamics of the Na+/K+-ATPase Probed by Voltage Clamp Fluorometry.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 964-969 (2003).</li><li>Lorenz, L., Pusch, M., Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heteromultimeric+CLC+chloride+channels+with+novel+properties.">Heteromultimeric CLC chloride channels with novel properties.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 13362-13366 (1996).</li><li>Mruk, K., Kobertz, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+Novel+Activator+of+KCNQ1-KCNE1+K++Channel+Complexes.">Discovery of a Novel Activator of KCNQ1-KCNE1 K+ Channel Complexes.</a> Biophys. J. 177a, (2009).</li><li>Kyte, J., Doolittle, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+diplaying+the+hydropathic+character+of+a+protein.">A method for diplaying the hydropathic character of a protein.</a> J. Mol. Biol. 157, 105-132 (1982).</li><li>Rakowski, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+movement+by+the+Na/K+pump+in+Xenopus+oocytes.">Charge movement by the Na/K pump in Xenopus oocytes.</a> J. Gen. Physiol. 101, 117-144 (1993).</li><li>Tsunoda, S. P., Hegemann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glu+87+of+Channelrhodopsin-1+Causes+pH-dependent+Color+Tuning+and+Fast+Photocurrent+Inactivation.">Glu 87 of Channelrhodopsin-1 Causes pH-dependent Color Tuning and Fast Photocurrent Inactivation.</a> Photochem. Photobiol. 85, 564-569 (2009).</li><li>Cha, A., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+implications+of+fluorescence+quenching+in+the+Shaker+K++channel.">Structural implications of fluorescence quenching in the Shaker K+ channel.</a> J. Gen. Physiol. 112, 391-391 (1998).</li><li>F&#246;rster, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ann Physik. 2, 55-75 (1948).</li><li>Dong, X., Stothard, P., Forsythe, I. J., Wishart, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PlasMapper:+a+web+server+for+drawing+and+auto-annotating+plasmid+maps.">PlasMapper: a web server for drawing and auto-annotating plasmid maps.</a> Nucleic Acids Res. 32, W660-W664 (2004).</li></ol>

Não há conflitos de interesse declarados.

A abordagem experimental que descrevemos combina site-specific rotulagem fluoróforo e duas de tensão eletrodo grampo para investigar a relação entre estrutura e função de proteínas da membrana. Esta técnica pode ser usada para obter tempo resolvido informações sobre a dinâmica conformacional de proteínas da membrana durante o transporte de íons. Além disso, esta abordagem pode ser adaptada para trabalhar com várias proteínas, tais como bombas de íons, canais iônicos e transportadores. Além de investigar a dinâmica de conformação de um resíduo específico, também é possível utilizar a transferência de energia de ressonância de fluorescência para determinar restrições de distância dentro de uma holoenzima. A determinação de distâncias entre os resíduos, bem como medir o movimento relativo entre subunidades pode ajudar a resolver questões fundamentais que envolvem mecanismos de propagação.

<table border="1"><tbody><tr><th> Reagente / Equipamentos </th><th> Companhia </th><th> Reagente / Equipamentos </th><th> Companhia </th></tr><tr><td> Fonte de Luz 100W Tungsten </td><td> Carl Zeiss microfilmagem </td><td> fluoresceína-5-maleimida </td><td> Invitrogen </td></tr><tr><td> 1B150F-4 </td><td> Precision Instruments mundo </td><td> Alto-Pure Kit de Extração PCR </td><td> Roche </td></tr><tr><td> 3,0-4,0 Ethicon Vicryl </td><td> Johnson &amp; Johnson </td><td> mMessage mMachine Kit </td><td> Ambion </td></tr><tr><td> 475AF40 filtro de excitação </td><td> Omega Optical </td><td> MS-222 </td><td> Sigma-Aldrich </td></tr><tr><td> 505DRLP dichroic espelho </td><td> Omega Optical </td><td> Nucleospin Kit Plasmid </td><td> Macherey-Nagel </td></tr><tr><td> Macherey-Nagel </td><td> Omega Optical </td><td> Nanodrop 2000c Sprectrophotometer </td><td> Thermo Scientific </td></tr><tr><td> 535DF50 filtro de excitação </td><td> Omega Optical </td><td> PC-10 Micropipeta Puller </td><td> Narishige </td></tr><tr><td> 560DRLP dichroic espelho </td><td> Omega Optical </td><td> pCLAMP 10 Software </td><td> Axon Instruments </td></tr><tr><td> 565ALP filtro de emissão </td><td> Omega Optical </td><td> PfuTurbo DNA polimerase </td><td> Strategene </td></tr><tr><td> 565EFLP filtro de emissão </td><td> Omega Optical </td><td> PIN-022A Photodiode </td><td> United Technologies Detector </td></tr><tr><td> 570DRLP dichroic espelho </td><td> Omega Optical </td><td> Filtros polarizados </td><td> Linos Phtonics Inc. </td></tr><tr><td> Linos Phtonics Inc. </td><td> Omega Optical </td><td> QuickChange Site-Directed Mutagenisis Kit </td><td> Stratagene </td></tr><tr><td> Axio Examiner microscópio de fluorescência </td><td> Carl Zeiss microfilmagem GMBH </td><td> RC-10 Câmara de fluorescência </td><td> Warner Instruments </td></tr><tr><td> Warner Instruments </td><td> Life Technologies </td><td> tetrametilrodamina-6-maleimida </td><td> Invitrogen </td></tr><tr><td> Digidata 1440A sistema de aquisição de dados </td><td> Axon Instruments </td><td> TOP10 Células Electrocompetent </td><td> Invitrogen </td></tr><tr><td> Dpn I </td><td> New England Biolabs </td><td> Turbo amplificador Tec-05X </td><td> npi </td></tr></tbody></table>

examining the conformational dynamics of membrane proteins in situ with site-directed fluorescence labeling

Dois eletrodos de voltagem clamp eletrofisiologia (TEVC) é uma ferramenta poderosa para investigar o mecanismo de ion transportes1 para uma grande variedade de proteínas da membrana incluindo canais iônicos 2, bombas de íons 3, 4 e transportadores. Os desenvolvimentos recentes têm combinado site-specific rotulagem fluoróforo ao lado TEVC, em simultâneo, analisar a dinâmica conformacional em resíduos específicos e função dessas proteínas na superfície de células individuais. Vamos descrever um método para estudar a dinâmica conformacional de proteínas da membrana, simultaneamente, o monitoramento de mudanças de fluorescência e de tensão atual usando clamp-fluorometria. Esta abordagem pode ser usada para examinar o movimento molecular de proteínas da membrana do local especificamente substituição cisteína seguinte e-site dirigido fluoróforo rotulagem 5,6. Além disso, este método fornece uma abordagem para determinar restrições de distância entre os resíduos específicos 7,8. Isto é conseguido através seletivamente anexando fluoróforos doador e receptor de dois resíduos de cisteína mutantes de interesse. Em suma, essas experiências são realizadas conforme expressão funcional da proteína desejada na superfície dos oócitos Xenopus Leavis. A grande área de superfície desses oócitos permite medições fáceis funcional e um sinal robusto fluorescência 5. Também é possível facilmente alterar as condições extracelulares como ligante pH, ou cátions / ânions, que podem fornecer informações adicionais sobre o mecanismo de proteínas da membrana 4. Finalmente, os desenvolvimentos recentes têm também permitiu a manipulação de selecionar íons interna após a co-expressão com uma segunda proteína 9. Nosso protocolo é descrito em várias partes. Primeiro, mutagênese cisteína digitalização passou por rotulagem fluoróforo é concluída em resíduos localizados na interface dos domínios transmembrana e extracelular. Experimentos subseqüentes são projetados para identificar resíduos que demonstram grandes mudanças na intensidade de fluorescência (&lt;5%) 3 em cima de uma mudança conformacional da proteína. Em segundo lugar, estas mudanças na intensidade de fluorescência são comparados com os parâmetros cinéticos da proteína de membrana, a fim de correlacionar a dinâmica conformacional para a função da proteína 10. Isto permite uma análise rigorosa biofísicos do movimento molecular da proteína-alvo. Por último, dois resíduos da holoenzima podem ser rotulados com um dador e receptor fluoróforo a fim de determinar restrições de distância usando métodos photodestruction doador. Também é possível monitorar o movimento relativo das subunidades da proteína seguintes rotulagem com um doador e aceitador fluoróforo.

Watch this Scientific Journal Video about Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling at JoVE.com

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

Vamos descrever um método que mede a cinética de transporte de íons de proteínas da membrana juntamente com site-specific análise das mudanças conformacionais usando fluorescência em células individuais. Esta técnica é adaptável a canais iônicos, transportadores e bombas de íons e pode ser utilizado para determinar restrições de distância entre as subunidades da proteína.

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 ...

examining-conformational-dynamics-membrane-proteins-situ-with-site

Research

JoVE Journal

Biology

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

14.5K visualizações.  Worcester Polytechnic Institute. Vamos descrever um método que mede a cinética de transporte de íons de proteínas da membrana juntamente com site-specific análise das mudanças conformacionais usando fluorescência em células individuais. Esta técnica é adaptável a canais iônicos, transportadores e bombas de íons e pode ser utilizado para determinar restrições de distância entre as subunidades da proteína.

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

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

Mutagênese do site caseiros Direção de Plasmids Whole

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Biologia

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 ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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 ...

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 ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

Examinando BCL-2 Função Família com grandes vesículas unilamelares

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Cryosectioning comunidades de leveduras para o exame de padrões de fluorescência

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

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 ...