Agradecemos a Beijing Wu para uso de seus dados. Este trabalho foi financiado por uma concessão de CARREIRA NSF para Jek

1. Preparação de ~ 50 nm de diâmetro pequenas vesículas Unilamelares (SUVs) para Folding proteína de membrana <ol><li> Uma solução de 1,2-dimiristoil-sn-glicero-3-fosfocolina (DMPC) lipídios em clorofórmio é comprado e aliquotado em frascos de vidro limpo em 20 mg por frasco para armazenamento de quantidades. A camada de gás nitrogênio é adicionado a cada frasco para evitar a oxidação lipídica, e os frascos são selados com tampões e parafilme. Os frascos são armazenados em um freezer -20 ° C até o uso. </li><li> Um único frasco que contém uma alíquota de 20 mg de lipídios em clorofórmio é usado para cada experimento. O conteúdo do frasco são secados usando um fluxo de gás nitrogênio por 1 hora até que não permanece solvente. </li><li> Lipídios secas são ressuspendidos em 1 mL de tampão fosfato 20 mM de potássio (pH = 7.3) usando um sonicador banho-maria durante ~ 30 segundos. Esta solução de lipídios suspenso aparecerá nublado, e é transferido para um tubo de 15 mL de plástico com um fundo cônico. Outra alíquota de 1 mL de tampão fosfato é adicionado ao frasco de vidro vazio que originalmente continha lipídios e sonicação é repetido, a solução é então adicionado ao tubo de 15 mL mesma. Este processo é repetido um total de 4 vezes até que o volume final no tubo é de 4 mL, resultando em uma concentração de lipídios de 5 mg / mL desta solução vesícula estoque. </li><li> O volume de 4 mL de solução vesícula lipídica é colocada num local quente (~ 30 ° C) banho de água e sonicado usando um MICROTIP ultrasonicator por 1 hora em ciclo de trabalho de 50%. A finalidade do banho de água é duplo. Primeiro, ela impede a solução de lipídios de tornar-se demasiado quente devido ao processo de sonicação. Segundo, o banho quente garante que a temperatura da solução aquosa de lipídios permanece acima da temperatura bicamada fase de transição durante sonicação; para DMPC, esta temperatura de transição é de aproximadamente 23 ° C. </li><li> A solução sonicado é passado através de uma seringa 0,22 mM de filtro para remover detritos da ponta sonicador, e esta solução é filtrada equilibrada durante a noite a 37 ° C. </li></ol> 2. Preparação de amostra para Curva de fluorescência inicial Unfolding <ol><li> A solução estoque de 10 M de uréia em 20 mM tampão fosfato (pH 7,3) é feita. É essencial que a água ser adicionado ao sólido (e não vice-versa), pois a grande quantidade de uréia ocupa um volume significativo na solução. Esta solução de uréia pode ser feita pela adição de solutos para uma garrafa limpa e vazia e adicionando água até ao volume final pretendido. Esta solução pode exigir o aquecimento em banho-maria ou em uma chapa quente, mexendo para dissolver o soluto. Uréia é higroscópico e, portanto, a concentração real da solução estoque de uréia deve ser determinada através do índice de refração 1. </li><li> Uma solução tampão estoque de 20 mM de fosfato (pH 7,3) é feita. </li><li> Uma solução estoque de proteína é desdobrada preparado. Esta solução de proteína de ações deve ter uma concentração de proteína M ~ 200 em 8 de uréia M, tampão fosfato 20 mM. </li><li> Amostras para estudos de fluorescência são preparados da seguinte maneira. Volumes adequados de proteína de ações (seção 2.3), a solução estoque de lipídeos (5 lipídica mg / mL, seção 1), estoque 10 solução de uréia M (seção 2.1) e tampão fosfato de estoque (seção 2.2) são combinados para fazer amostras contendo ~ 4 proteína M, 1 mg / mL de lipídios, e 0-8 M de uréia em incrementos de 1 M. O volume total de cada amostra é de 200 mL. Uma planilha exemplo (Tabela 1) está incluído o que volumes listas exigidas usando uma solução de proteína M 200 ações. </li><li> Amostras em branco são feitas conforme descrito na seção 2.4, no entanto, a proteína não é adicionado. No lugar de proteína, 4 mL de uma solução de uréia 8 M podem ser adicionados para as concentrações de outros componentes são idênticos aos do ponto 2.4. Estes espaços são utilizados para subtrair espalhamento e sinal de fundo outra que aparece nos espectros de fluorescência da proteína. </li><li> Amostras de seções 2.4 e 2.5 são autorizados a incubar a 37 ° C por pelo menos 2 horas antes da medição de espectros de fluorescência para garantir dobrar completa e equilíbrio. </li></ol> 3. Medição de espectros de fluorescência Triptofano fluorescência de cada amostra e em branco é medida usando um fluorímetro steady-state. Espectros de fluorescência deve ser gravado com comprimento de onda de excitação de 290 nm para evitar a excitação de resíduos de tirosina, e digitalizados 305-500 nm. Entrada típica e bandpass de saída é de 3 nm. O incremento de comprimento de onda e tempo de integração pode ser otimizada para sinal-ruído. Valores típicos para o incremento de comprimento de onda e tempo de integração são 1 nm / etapa e 0,5 seg / step, respectivamente. Membrana proteínas em geral dobrar em lipídios sintéticos somente quando a temperatura está acima da temperatura bicamada fase de transição. Portanto, neste caso de DMPC, a temperatura da amostra é mantida constante em 30 ° C. A microvolume cubeta de sílica fundida com capacidade de 160 mL é utilizado nestes exexperimentos. 4. Geração de Curve Unfolding inicial e Estimativa da energia livre de Gibbs da proteína de membrana Unfolding <ol><li> Espectros de fluorescência na forma de um conjunto de dados xy são carregados no software, tais como Igor Pro, Matlab, Origin, ou Excel. </li><li> Sinal do fundo da vesícula uréia e lipídeos deve ser subtraído utilizando o seguinte procedimento: Spectrum A = espectro de fluorescência-prima de proteína na presença de vesículas lipídicas e de concentração específicos de uréia. B = espectro espectro primas fluorescência de amostra em branco correspondente que contém vesículas lipídicas e concentração de uréia mesma que no espectro de A; nenhuma proteína nas amostras está em branco. Spectrum Spectrum corrigido = A - C * espectro B. C é um escalar normalmente igual a 1,0. Em alguns casos, C é menor ou maior que 1.0 por causa de problemas de dispersão que causam sobre-ou sub-subtração perto da região nm ~ 305-320. </li><li> O comprimento de onda de máxima de fluorescência, λ max, é tabulados a partir de espectros corrigidos para cada concentração de uréia. Um valor típico para a plena desdobrado proteína de membrana é de 350 nm, enquanto a de proteína dobrada é de aproximadamente 330 nm. Comprimentos de onda tabelados são convertidos em fração desdobrado convertendo o intervalo de valores λ max (~ 330 a ~ 350 nm) para a faixa 0,0-1,0 correlacionar a λ valor máximo a fração da população se desenrolava. Por exemplo, um valor máximo de λ 330 nm corresponde a 0,0, a 350 nm corresponde a 1,0, e 340 nm corresponde a 0,5 em termos de fração desdobrado. Esta fração de proteína é, então, desdobrado plotado contra a concentração de uréia. Como dito acima, a concentração de uréia deve ser determinada experimentalmente através da medição do índice de refração. </li><li> Supomos que a proteína pode existir em um dos dois estados: dobrado ou desdobrado. O enredo da fração desdobrado, f, versus concentração de uréia, C, pode estar em forma para a equação: 2 <img src="/files/ftp_upload/2669/2669eq2.jpg" alt="Equação 2" /> O coeficiente de m corresponde à taxa de variação da energia livre com respeito à concentração desnaturante e C m corresponde à concentração de uréia no ponto médio em que a população dobrada é igual a população se desenrolava. Estes valores são as variáveis ​​encaixe obtido a partir de ajuste dos mínimos quadrados por dados software de análise. R (0.001987 kcal / mol • K) é a lei do gás constante, e T é a temperatura (303 K). </li><li> Os coeficientes obtidos a partir do ajuste são utilizadas para determinar a energia livre de Gibbs (kcal / mol) de desdobramento, na ausência de uréia: <img src="/files/ftp_upload/2669/2669eq3.jpg" alt="Equação 3"/> . </li></ol> 5. Otimizado Unfolding Curva para medições mais precisas de energia livre de Gibbs de desdobramento. <ol><li> A curva de desdobramento descrito acima revela a gama de concentrações de uréia que faz com que a maior mudança no λ max. Esta gama é normalmente pequeno, ea maioria de desdobramento ocorre dentro desta faixa de pequeno porte. , A fim de determinar com precisão a energia livre de desdobramento, amostras adicionais são analisados ​​nesta região para obter um lote que contém muitos pontos dados necessários para a montagem dos mínimos quadrados otimizado. Por exemplo, se a proteína se desdobra entre 2 e 4 de uréia M, as amostras podem ser feitas que contêm 2-4 M de uréia em incrementos de 0,2 M para revelar mais precisamente a forma desta importante região da curva de desenvolvimento. Não é necessário medir espectros em branco em cada concentração de uréia, é suficiente para medir um espectro em branco a cada passo ~ 0,5 M. </li><li> A energia livre de desdobramento obtido a partir deste enredo pode ser ligeiramente diferente do obtido na curva original desdobramento, mas reflete um valor mais preciso. </li></ol> 6. Resultados representativos: <table border="1"><tbody><tr><td> Amostra </td><td> final [uréia], M </td><td> proteína de ações (mL) </td><td> lipídica de ações (mL) </td><td> estoque 10 M uréia (mL) </td><td> estoque regulador (mL) </td></tr><tr><td> 1 </td><td> ~ 0,16 </td><td> 4 </td><td> 40 </td><td> 0 </td><td> 156 </td></tr><tr><td> 2 </td><td> 1 </td><td> 4 </td><td> 40 </td><td> 20 </td><td> 136 </td></tr><tr><td> 3 </td><td> 2 </td><td> 4 </td><td> 40 </td><td> 40 </td><td> 116 </td></tr><tr><td> 4 </td><td> 3 </td><td> 4 </td><td> 40 </td><td> 60 </td><td> 96 </td></tr><tr><td> 5 </td><td> 4 </td><td> 4 </td><td> 40 </td><td> 80 </td><td> 76 </td></tr><tr><td> 6 </td><td> 5 </td><td> 4 </td><td> 40 </td><td> 100 </td><td> 56 </td></tr><tr><td> 7 </td><td> 6 </td><td> 4 </td><td> 40 </td><td> 120 </td><td> 36 </td></tr><tr><td> 8 </td><td> 7 </td><td> 4 </td><td> 40 </td><td> 140 </td><td> 16 </td></tr></tbody></table> Tabela 1. Volume das soluções de reserva necessária para fazer amostras de fluorescência <img src="/files/ftp_upload/2669/2669fig1.jpg" alt="Figura 1" /> Figura 1. Triptofano espectros de fluorescência de ~ 5 proteína de membrana M representante que contém um resíduo de triptofano único. (A) Espectros de fluorescência da proteína Raw (Um espectro de 4,2), (B) Raw espectros de fluorescência de branco (espectro B de 4,2), (C) Corrigido espectro de 4,2. <img src="/files/ftp_upload/2669/2669fig2.jpg" alt="Figura 2" /> Figura 2. Corrigida triptofano espectros de fluorescência da proteína de membrana a partir da Figura 1 para as concentrações de uréia numerosos. <img src="/files/ftp_upload/2669/2669fig3.jpg" alt="Figura 3" /> Figura 3. Unfolding curva obtida a partir de dados na Figura 2, com ajuste para a equação em 4,4. A energia livre de Gibbs é calculado de acordo com a secção 4.5.

<ol>
	<li>Shirley, B. A., Shirley, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Folding and Stability. , 177-190 (1995).</li><li>Pace, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+and+Analysis+of+Urea+and+Guanidine+Hydrochloride+Denaturation+Curves.">Determination and Analysis of Urea and Guanidine Hydrochloride Denaturation Curves.</a> Methods Enzymol. 131, 266-279 (1986).</li><li>Lau, F. W., Bowie, J. U. <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+Assessing+the+Stability+of+a+Membrane+Protein.">A Method for Assessing the Stability of a Membrane Protein.</a> Biochemistry. 36, 5884-5892 (1997).</li><li>Burgess, N. K., Dao, T. P., Stanley, A. M., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;-Barrel+Proteins+that+Reside+in+the+Escherichia+coli+Outer+Membrane+in+Vivo+Demonstrate+Varied+Folding+Behavior+in+Vitro.">&#946;-Barrel Proteins that Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro.</a> J. Biol. Chem. 283, 26748-26758 (2008).</li><li>Booth, P. J., Curnow, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+scene+investigation:+membrane+proteins.">Folding scene investigation: membrane proteins.</a> Curr. Opin. Struct. Biol. 19, 8-13 (2009).</li><li>Hong, H., Tamm, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+coupling+of+integral+membrane+protein+stability+to+lipid+bilayer+forces.">Elastic coupling of integral membrane protein stability to lipid bilayer forces.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 4065-4070 (2004).</li><li>Sanchez, K. M., Gable, J. E., Schlamadinger, D. E., Kim, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Tryptophan+Microenvironment,+Soluble+Domain,+and+Vesicle+Size+on+the+Thermodynamics+of+Membrane+Protein+Folding:+Lessons+from+the+Transmembrane+Protein+OmpA.">Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA.</a> Biochemistry. 47, 12844-12852 (2008).</li></ol>

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O protocolo atual descreve a geração de curvas de desdobramento da membrana-associados proteínas e peptídeos que contêm resíduos de triptofano. Aqui, presume-se que a fluorescência de triptofano reflete se a proteína é dobrado e inserido em vesículas lipídicas sintéticas, ou desdobrado em solução. Suposições adicionais, tais como dois estados dobrar e dependência linear de energia livre com a concentração de desnaturante, são feitas no presente relatório;. Modificação destes pressupostos resultado...

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thermodynamics of membrane protein folding measured by fluorescence spectroscopy

Enovelamento de proteínas da membrana é um tema emergente com significado fundamental e saúde. A abundância de proteínas da membrana das células está subjacente a necessidade de estudo detalhado da dobragem desta família de proteínas onipresente. Além disso, os avanços na nossa capacidade de caracterizar doenças associadas com proteínas deformadas ter motivado significativos esforços experimentais e teóricos no campo de enovelamento de proteínas. Progresso rápido neste domínio importante é, infelizmente, dificultada pela desafios inerentes associados com proteínas da membrana e da complexidade do mecanismo de dobramento. Aqui, descrevemos um procedimento experimental para medir a propriedade termodinâmica da energia livre de Gibbs de desdobramento, na ausência de desnaturante, Δ G ° H 2 O, por uma proteína de membrana representante integrante da E. coli. Este protocolo incide sobre a aplicação da espectroscopia de fluorescência para determinar as populações dos estados de equilíbrio dobrada e desdobrada em função da concentração de desnaturante. Considerações experimentais para a preparação de vesículas lipídicas sintéticas, bem como etapas fundamentais no processo de análise de dados são realçados. Esta técnica é versátil e pode ser perseguido com diferentes tipos de desnaturante, incluindo temperatura e pH, bem como em vários ambientes de dobramento de lipídios e micelas. O protocolo atual é aquele que pode ser generalizada para qualquer membrana ou proteína solúvel que atenda o conjunto de critérios discutidos abaixo.

Watch this Scientific Journal Video about Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy at JoVE.com

Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Este artigo detalha o procedimento de vídeo experimental para a obtenção da energia livre de Gibbs da dobradura de proteína de membrana por fluorescência de triptofano.

Termodinâmica de enovelamento de proteínas de membrana Medido por espectroscopia de fluorescência

Membrane protein folding is an emerging topic with both fundamental and health-related significance.  The abundance of membrane proteins in cells ...

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18.2K visualizações.  University of California San Diego - UCSD. Este artigo detalha o procedimento de vídeo experimental para a obtenção da energia livre de Gibbs da dobradura de proteína de membrana por fluorescência de triptofano.

Vídeo: Thermodynamics of Membrane Protein Folding Measured by Fluorescence Spectroscopy

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

Métodos de detecção de fluorescência para as plataformas de gotículas microfluídicos

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengenharia

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Misturadores microfluídicos para Estudar o dobramento de proteínas

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to identify and characterize their interactions are necessary5. To this end, we developed the Membrane Strep-protein interaction experiment, called Membrane-SPINE6. This technique combines in vivo cross-linking using the reversible cross-linker formaldehyde with affinity purification of a Strep-tagged membrane bait protein. During the procedure, cross-linked prey proteins are co-purified with the membrane bait protein and subsequently separated by boiling. Hence, two major tasks can be executed when analyzing protein-protein interactions (PPIs) of membrane proteins using Membrane-SPINE: first, the confirmation of a proposed interaction partner by immunoblotting, and second, the identification of new interaction partners by mass spectrometry analysis. Moreover, even low affinity, transient PPIs are detectable by this technique. Finally, Membrane-SPINE is adaptable to almost any cell type, making it applicable as a powerful screening tool to identify PPIs of membrane proteins.

Membrane-SPINE: A Biochemical Tool to Identify Protein-protein Interactions of Membrane Proteins In Vivo

A biochemical approach is described to identify in vivo protein-protein interactions (PPI) of membrane proteins. The method combines protein cross-linking, affinity purification and mass spectrometry, and is adaptable to almost any cell type or organism. With this approach, even the identification of transient PPIs becomes possible.

Membrana-COLUNA: Uma Ferramenta bioquímicos na identificação de interações proteína-proteína de Proteínas de Membrana<em&gt; In Vivo</em

Membrane proteins are essential for cell viability and are therefore important therapeutic targets1-3. Since they function in complexes4, methods to ...

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

De alta resolução espacial e temporal Análise de Receptor Dynamics por uma única molécula de microscopia de fluorescência

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope1. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns2. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam3-5. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell6,7. Cells were plated according to standard protocols, and stained using a commercially lysosome dye. We discuss briefly the hardware configuration and in more detail the control software, to perform a 3D orbital tracking experiment inside living cells. We discuss in detail the parameters required in order to control the scanning microscope and enable the motion of the beam in a closed orbit around the particle. We conclude by demonstrating how this method can be effectively used to track the fast motion of a labeled lysosome along microtubules in 3D within a live cell. Lysosomes can move with speeds in the range of 0.4-0.5 &#181;m/sec, typically displaying a directed motion along the microtubule network8.

In this video protocol we track - at high speed and in three dimensions - fluorescently labeled lysosomes within living cells, using the orbital tracking method in a modified two-photon microscope.

3D Orbital Rastreamento em um microscópio de dois fotões Modificação: Uma Aplicação para o rastreamento de vesículas intracelulares

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparação de Matriz Extracelular fibras de proteína de Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Engenharia 'Dourada' fluorescência por pressão seletiva de incorporação de aminoácidos não-canônicos e análise de proteínas por espectrometria de massa e fluorescência

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Deteção da endotoxina em Nano-formulações usando Limulus Amoebocyte Lysate (LAL) ensaios

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Na síntese de Vesiculo de precursores de membrana peptídica para crescimento de vesículas autônomas

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

Molecular Spring Constant Analysis by Biomembrane Force Probe Spectroscopy

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure single-molecular binding kinetics, assess mechanical properties of ligand-receptor interactions, visualize protein dynamic conformational changes and more excitingly elucidate receptor mediated cell mechanosensing mechanisms. More recently, BFP has been used to measure the spring constant of molecular bonds. This protocol describes the step-by-step procedure to perform molecular spring constant DFS analysis. Specifically, two BFP operation modes are discussed, namely the Bead-Cell and Bead-Bead modes. This protocol focuses on deriving spring constants of the molecular bond and cell from DFS raw data.

A biomembrane force probe (BFP) is an in situ dynamic force spectroscopy (DFS) technique. BFP can be used to measure the spring constant of molecular interactions on living cells. This protocol presents spring constant analysis for molecular bonds detected by BFP.

Análise constante da mola molecular por espectroscopia da sonda de força biomembrana

A biomembrane force probe (BFP) has recently emerged as a native-cell-surface or in situ dynamic force spectroscopy (DFS) nanotool that can measure ...