The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1. Andaime lipossomas Preparação NOTA: Idealmente, as experiências iniciais deve usar um andaime relativamente simples e sem traços característicos (composto de DOPC (1,2-dioleoyl--sn-glicero-3-fosfocolina ou PC) e DGS-NTA (Ni 2+) (1,2-dioleoil - sn-glicero-3 - [(N - (5-amino-1-carboxipentil) ácido iminodiacético) succinil]. (sal de níquel)) edifício fora destas experiências, a carga de lípidos, a flexibilidade, e a curvatura pode ser introduzido como factores individuais com o potencial para alterar as interacções da membrana proximal. Estas alterações podem ser conseguida por adição de quantidades definidas de componentes lípidos específicos, incluindo fosfatidilserina ou cardiolipina (CL), f osf atidiletanolamina (DOPE ou PE), ou galactosil (β) ceramida. <ol><li> Combinar lipidos dissolvidos em clorofórmio em um tubo de ensaio de vidro limpo. Evapora-se o solvente com azoto gasoso seco durante a rotação do tubo de modo a formar uma película fina de lípidos. Remover o solvente residual com uma centrifugal evaporador durante 1 h a 37 ° C. NOTA: As várias formulações lipossomais são usadas nos protocolos descritos abaixo: lipossomas de andaime (3,3 mol% DGS-NTA (Ni 2+) / 96,7% molar de DOPC), lipossomas de andaime com cardiolipina (3,3 mol% DGS-NTA (Ni 2+) / 10% molar de cardiolipina / 86,7% molar de DOPC), lipossomas de andaime flexíveis com cardiolipina (3,3 mol% DGS-NTA (Ni 2+) / 10% molar de cardiolipina / 35% molar de DOPE / DOPC 51,7 mol%), e de andaime enriquecido lipossomas (10 mol% DGS-NTA (Ni 2+) / 15% molar de cardiolipina / 35% molar de DOPE / 40% molar de DOPC). </li><li> Adicionar tampão A (25 mM de HEPES (ácido 4- (2-hidroxietil) -1-piperazinoetanossulfónico), KCl 150 mM, pH ajustado para 7,5 com KOH) pré-aquecido a 37 ° C, de modo que a concentração final de lípido é de 1 - 2 mm. Incubar 30 min a 37 ° C com agitação em vórtice para ressuspender completamente ocasional da mistura de lípidos (Figura 1a). </li><li> Transferir para um tubo de ensaio de plástico, colocar o tubo em nitrogênio líquido até que esteja completamente congelado (rougHly 30 s), e coloque em uma temperatura de 37 ° C banho de água até estar totalmente descongelado (cerca de 1-2 min). Repetir para um total de 4 ciclos de congelação-descongelação (Figura 1B). </li><li> Prepara-se uma extrusora de lípido por imersão 4 suportes de filtro e um filtro de policarbonato e em tampão de montagem da extrusora de acordo com as instruções do fabricante. Extrudir a solução de lípidos através do filtro 21 vezes. Use pressão suave e constante para assegurar uma distribuição de tamanho homogêneo (Figura 1c). NOTA: Para todas as experiências descritas neste protocolo, um filtro de 1,0 um policarbonato foi usada para a extrusão. Drp1 interacção com lípidos aniónicos pode ser observada com uma variedade de diâmetros de lipossomas variando desde 50 nm a 400 nm, 12 ou maior 13. Por isso, o tamanho do filtro de 1 ^ M foi escolhida para ser ideal, tanto para a actividade de GTPase e por microscopia electrónica. Se outros diâmetros de lipossomas são desejados, preparação de gigantes vesículas unilamelares 14,15 (GUVs) ou pequena unilamellAR vesículas 16 (SUVs) pode ser usado. Dispersão dinâmica de luz pode ser utilizado para avaliar o tamanho do lipossoma heterogeneidade 13. </li><li> Armazenar lipossomas extrudidos a 4 ° C e depois descartá 3-5 d. </li></ol> 2. Uso de andaime lipossomas para Análise de ligação às proteínas <ol><li> Preparação de amostra <ol><li> Incubar marcada com His MffΔTM (5 uM final), com lipossomas de andaime (40 mol% de PC / 35% molar de PE / mole 15% CL / 10% molar de SGD-NTA (Ni 2+), 50 uM final) durante pelo menos 15 min à TA em Tampão a + BME (25 mM de HEPES, 150 mM de KCl, 10 mM de β-mercaptoetanol (BME), pH ajustado para 7,5 com KOH). Para um controle Mff-livre, incubar lipossomas com uma marcada com His proteína de controlo (tal como GFP) para ligar e escudo exposta NTA (Ni 2+). NOTA: MffΔTM foi expressa e purificada como descrito em um estudo prévio 2. GFP foi purificado de uma maneira similar, mas o passo de permuta iónica foi omitido. BME foi necessária para estes experimenTS porque Drp1 é sensível à oxidação, o qual pode alterar as suas propriedades de actividade e de montagem. </li><li> Adicionar Drp1 (2 uM final) e incubar durante 1 h a RT. NOTA: Drp1 foi expressa e purificada como descrito em um estudo prévio 2. Após incubação com Drp1, o efeito de deformação de ligação de membrana de nucleótidos pode ser investigada através da incubação de uma hora adicional com 2 mM de MgCl2 e 1 mM de GTP, quer, 1 mM de GMP-PCP, ou tampão A + BME. </li></ol></li><li> Negativo Stain Transmission Electron Microscopy (EM) Análise <ol><li> Transferência de 5 uL de amostra a uma folha de película de laboratório, e estabelecer uma rede de Cu / Rh revestido-carbono sobre a amostra. Incubar a grade 1 min sobre a amostra, limpar o excesso de líquido em papel de filtro, e transferir para uma queda de 2% de acetato de uranilo. Incubar 1 min, blot excesso de corante em papel de filtro, e transferir para um caixa de grade. Loja sob vácuo O / N para assegurar a dessecação completa. </li><li> amostras de imagem usando um microsc eletrônica de transmissãoope em 18.500 - 30,000X ampliação para observar as alterações ultra-estruturais em proteínas e lipossomas morfologias 17. Nota: As alterações ultra-estruturais podem ser quantificados usando software de análise de imagem, tais como ImageJ 13 (http://imagej.nih.gov/ij/). decoração proteína pode ser medido quando comparada com modelos nus lipídicas. Além disso, os diâmetros dos segmentos tubulares pode ser medido a partir da porção mais externa dos conjuntos 13. Uma análise mais detalhada pode ser realizada através de microscopia crio-eletrônico de 17. Este método pode ser utilizado para os conjuntos de proteínas em lípidos nativas imagem em solvente, sem a utilização de manchas de metais pesados ​​que o revestimento da amostra. Desta forma, as características estruturais detalhados não aparentes na coloração negativa, incluindo mudanças na morfologia lipídico subjacente pode ser examinados e quantificados. </li></ol></li></ol> 3. Uso de andaime lipossomas para Ensaio Enzimático Nota: A coloriensaio GTPase métrica 18 foi utilizado para medir a libertação de fosfato por hidrólise GTP. GTPase ensaios alternativos estão disponíveis 19 e pode ser implementado conforme necessário. <ol><li> Incubar marcada com His MffΔTM (QFP), Fis1ΔTM (Fis1), ou GFP (5 uM final para todos) com lipossomas de andaime (150 uM final) durante 15 min à temperatura ambiente em Tampão A + BME (volume = 30 mL). Adicionar Drp1 (500 nM final) e incuba-se um adicional de 15 min à temperatura ambiente (= 80 mL de volume). NOTA: Fis1 foi purificado de uma maneira similar à Mff 2, mas o passo de cromatografia de permuta iónica foi omitido. O propósito de Sua marcado com GFP é para proteger o NTA (Ni 2+) headgroups e evitar interacções de carga não específicas com outras proteínas. Se nenhum efeito é observado na ausência de GFP, então pode não ser necessária este controlo. proteínas alternativos de bloqueio (de tamanho comparável à da proteína de interesse) pode ser utilizado, bem como, mas GFP permite a visualização directa das interacções com SCAFdobre lipossomas. </li><li> Tubos de transferência para um termociclador definido como 37 ° C, e iniciar as reacções por adição de GTP e MgCl2 (1 mM e final de 2 mM, respectivamente; volume = 120 mL). </li><li> Em pontos de tempo desejado (isto é, T = 5, 10, 20, 40, 60 min), a transferência de 20 uL da reacção aos poços de uma placa de microtitulação contendo 5 uL de EDTA 0,5 M para quelar Mg 2+ e parar a reacção. </li><li> Preparar um conjunto de padrões através da diluição de fosfato de KH 2 PO 4 em tampão A + BME para calibrar os resultados. Um conjunto útil de padrões é de 100, 80, 60, 40, 20, 10, 5 e 0 uM. Adicionar 20 ul de cada uma a poços contendo 5 uL de 0,5 M de EDTA. </li><li> Adicionar 150 uL de reagente de malaquite verde (1 mM de malaquite verde carbinol, 10 mM de molibdato de amónio tetrahidratado, e HCl 1 N) a cada alvéolo, e leu OD650 5 min após a adição. NOTA: GTP é ácido lábil, e irão hidrolisar na presença de reagente de verde de malaquite. Certifique-se que tele o tempo entre a adição do reagente malaquita e leitura é constante para garantir a reprodutibilidade dos resultados. </li><li> Gerar uma curva padrão através da representação gráfica OD 650 dos padrões como uma função da concentração de fosfato. Use de regressão linear para determinar a relação entre a OD 650 e a concentração de fosfato numa amostra. </li><li> Usando a regressão linear, converter a OD 650 das amostras de reacção de fosfato de proteína para uM. Determinar a taxa de geração de fosfato para cada mistura de reacção através da representação gráfica concentração de fosfato como uma função do tempo, e converter a k cat dividindo a taxa pela concentração Drp1 (0,5 uM). NOTA: Apenas a taxa linear inicial deve ser utilizado para determinar a taxa de geração de fosfato, e um mínimo de 3 pontos de dados devem ser usados. Se a velocidade de reacção é suficientemente rápida que os três primeiros pontos de dados não é linear (isto é, o R2 do ajuste linear é inferior a 0,9) um sinalcurso de tempo mais curto ificantly com, pelo menos, 3 pontos de tempo deve ser realizada. </li></ol>

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The authors have nothing to disclose.

Este protocolo oferece um método para a investigação das interacções proteína-proteína envolvendo proteínas de membrana integrais. Através da utilização de um lipossoma andaime modular, os investigadores são capazes de avaliar a actividade de uma ou mais proteínas num ambiente de lípido-proximal. Estudos anteriores têm demonstrado um método semelhante por enzimas de receptor da membrana do plasma 24-26. Nós expandimos esse método para incorporar cofatores lipídicos e e...

Estudando interações proteína-proteína de membrana-proximal é uma tarefa desafiadora devido à dificuldade de recapitular o ambiente nativo das proteínas de membrana integrais envolvidas 1. Isto é devido à necessidade de solubilização detergente e a orientação inconsistente de proteínas em proteolipossomas. A fim de evitar estes problemas, que têm empregue uma estratégia em que os domínios solúveis de proteínas de membrana integrais são expressos como proteínas de fusão His-tag, e estes fragmentos solúveis estão ancoradas aos lipossomas através de interacções de andaime com NTA (Ni 2+) headgroups no lípido superfície. Usando estes andaimes, as interacções proteína-lipídicos proximal pode ser investigada através de uma gama de composições de lípidos e proteínas. Temos eficazmente aplicado esse método para investigar as interacções proteína-proteína crítica que governam a montagem do complexo de fissão mitocondrial e examinar interacções lípidos que modulam este processo 2. Durante fissão mitocondrial, uma proteína de remodelação da membrana conservada, chamada proteína relacionada com dinamina 1 (Drp1) 3, é recrutado para a superfície do mitocondrial externa da membrana (OMM) em resposta a sinais celulares que regulam a homeostase de energia, sinalização apoptótica, e vários outros processos mitocondriais integral. Esta grande GTPase, citosólica é recrutada para a superfície das mitocôndrias através de interacções com proteínas integrais OMM 4 - 8. O papel de uma tal proteína, Cisão do Factor mitocondrial (QFP), tem sido difícil para elucidar devido a uma interacção fraca aparente com Drp1 in vitro. No entanto, estudos genéticos demonstraram claramente que Mff é essencial para o êxito da fissão 7,8 mitocondrial. O método descrito neste manuscrito foi capaz de superar as deficiências anteriores, introduzindo interações lipídicas simultâneas que promovem interações Drp1-QFP. No geral, este romance REVEA ensaiolevou interações fundamentais que orientam a montagem do complexo de fissão mitocondrial e forneceu uma nova etapa para estudos estruturais e funcionais em curso desta máquina molecular essencial. Até à data, a análise de interacções entre Drp1 e Mff ter sido complicada pela flexibilidade inerente do Mff 9, a heterogeneidade dos polímeros Drp1 2,10, e a dificuldade de purificação e a reconstituição de comprimento completo Mff com um domínio transmembranar de 11 intacta. Nós abordado esses desafios usando lipossomas NTA (Ni 2+) de andaime para reconstituir marcada com His Mff faltando seu domínio transmembranar (MffΔTM-His 6). Esta estratégia foi vantajoso porque MffΔTM foi extremamente solúvel quando sobre-expresso em E. coli, e esta proteína foi isolado facilmente reconstituída em lipossomas de andaime. Quando preso a estes modelos de lípidos, Mff assumido um, voltados para o exterior orientação idêntica na superfície da membrana.Além destas vantagens, lípidos mitocondriais, tais como a cardiolipina, foram adicionados para estabilizar Mff dobragem e de associação com a membrana 11. A cardiolipina também interage com o domínio variável de 2,12 Drp1 que podem estabilizar esta região desordenada e facilitar a montagem da máquina de fissão. Este método robusto é amplamente aplicável para futuros estudos que visam avaliar as interações proteína de membrana-proximal. Através do uso de interacções amarrar / afinidade adicionais, a sofisticação destes estudos de reconstituição da membrana pode ser aumentada para imitar complexidade adicional encontrado na superfície das membranas dentro das células. Ao mesmo tempo, as composições de lípidos podem ser modificados para mimetizar mais precisamente os ambientes nativas destes complexos macromoleculares. Em resumo, este método proporciona um meio para examinar as contribuições relativas de proteínas e lípidos em moldar a morfologias de membrana durante Proc celulares críticascessos.

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using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

Estudos de proteínas de membrana integrais in vitro são frequentemente complicada pela presença de um domínio transmembranar hidrófobo. complicando ainda mais estes estudos, a reincorporação de proteínas de membrana de detergente-solubilizado em lipossomas é um processo estocástico, onde topologia proteína é impossível de aplicar. Este documento apresenta um método alternativo para estas técnicas difíceis que utiliza uma estrutura de suporte à base de lipossomas. a solubilidade da proteína é aumentada pela supressão do domínio transmembranar, e estes aminoácidos são substituídos com um radical amarrar, tal como uma cauda de His. Este tirante interage com um grupo de ancoragem (Ni2 + coordenado por ácido nitrilotriacético (NTA (Ni 2+)) para proteínas marcadas com His), que executa uma topologia uniforme de proteína na superfície do lipossoma. Um exemplo é apresentado, em que a interacção entre a proteína relacionada com um dinamina (Drp1) com uma proteína de membrana integral, Cisão do Factor mitocondrial (QFP), foi investigated usando este método scaffold lipossoma. Neste trabalho, nós demonstramos a capacidade de recrutar Mff eficientemente Drp1 solúvel à superfície dos lipossomas, o que estimulou a sua actividade de GTPase. Além disso, foi capaz de Drp1 tubulate o modelo lípido Mff-decorado na presença de lípidos específicos. Este exemplo demonstra a eficácia de lipossomas de andaime usando ensaios estruturais e funcionais e destaca o papel do QFP, a regulação da actividade Drp1.

Embora a interacção entre Drp1 Mff e foi demonstrada ser importante para a fissão mitocondrial, esta interacção tem sido difícil de recapitular in vitro. Nosso objetivo era melhor emular o ambiente celular no qual Drp1 e do QFP interagir. Para este fim, os lipossomas contendo concentrações limitativos de NTA (Ni 2+) headgroups foram preparados através da hidratação de uma película de lípido, tal como descrito acima. A solução de lípido consiste inicial...

Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Usando andaime lipossomas para reconstituir interações proteína-proteína Lipid-proximal<em&gt; In Vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

using-scaffold-liposomes-to-reconstitute-lipid-proximal-protein

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

8.7K visualizações.  Case Western Reserve University School of Medicine. This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment. 

Vídeo: Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

Anisotropia de fluorescência como uma ferramenta para estudar as interações proteína-proteína

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

Bioquímica

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

resolvida no tempo com ionização por electrospray Hidrogénio-deutério troca Espectrometria de Massa para estudar a estrutura e dinâmica das proteínas

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

Peptídeos biotinylated célula-penetrante para estudar interações da proteína-proteína intracelular

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Usando três cores único-molécula FRET para estudar a correlação de interações da proteína

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

Manipulando células vivas para construir estável montagem celular 3D sem andaime Artificial

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

Produção em grande escala de RNAs recombinantes em um andaime Circular usando um sistema derivado de Viroid em Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Um ensaio de espectroscopia de flutuação de fluorescência das interações da proteína-proteína em contatos do celular

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Mutagenesis local-dirigido para In Vitro e In Vivo experimentos exemplificados com interações de RNA em Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

Aplicação de interferometria Biolayer (BLI) para o estudo de interações proteína-proteína em transcrição

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Avaliação de interações proteína-proteína usando uma técnica de digestão na membrana

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...