Damos las gracias a Beijing Wu para el uso de sus datos. Este trabajo fue apoyado por un premio CARRERA NSF a JEK

1. Preparación de ~ 50 nm de diámetro pequeño vesículas unilaminares (SUV) de plegado de proteínas de membrana <ol><li> Una solución de 1,2-dimiristoil-sn-glicero-3-fosfocolina (DMPC) lípidos en cloroformo se compra y alícuotas en viales de vidrio limpia en cantidades de 20 mg por vial para el almacenamiento. Una capa de gas nitrógeno, se añade a cada vial para evitar la oxidación de lípidos, y los viales están sellados con tapas y parafina. Los viales se almacenan en un congelador a -20 ° C hasta su uso. </li><li> Un solo frasco que contiene una alícuota de 20 mg de lípidos en cloroformo se utiliza para cada experimento. El contenido de la ampolla se seca con una corriente de gas nitrógeno durante 1 hora hasta que ya no sigue siendo solvente. </li><li> Lípidos secos se resuspendieron en 1 ml de 20 mM de tampón fosfato de potasio (pH = 7,3) con un baño de ultrasonido de agua para aproximadamente 30 segundos. Esta solución de lípidos suspendido aparecerá nublado, y se transfiere a un plástico de 15 ml del tubo con un fondo cónico. Otra alícuota de 1 ml de solución amortiguadora de fosfato se añade a la ampolla de vidrio vacía que contenía originalmente los lípidos, y sonicación se repite, la solución se añade a la misma de 15 ml tubo. Este proceso se repite un total de 4 veces hasta que el volumen final en el tubo es de 4 ml, resultando en una concentración de lípidos de 5 mg / mL de esta solución de vesículas de valores. </li><li> El volumen de 4 ml de solución de lípidos vesícula se coloca en un lugar cálido (30 ° C) baño de agua, y se sonicaron utilizando un micropunta ultrasonicador durante 1 hora a 50% del ciclo. El propósito del baño de agua es doble. En primer lugar, evita que la solución de lípidos se vuelva demasiado caliente debido al proceso de sonicación. En segundo lugar, el baño caliente asegura que la temperatura de la solución acuosa de lípidos se mantiene por encima de la temperatura durante la fase de transición de dos capas sonicación, por DMPC, esta temperatura de transición es de ~ 23 ° C. </li><li> La solución sonicado se pasa a través de una jeringa de 0,22 micras filtro para eliminar los restos de la punta sonicador, y esta solución filtrada se equilibra la noche a 37 ° C. </li></ol> 2. Preparación de muestras para la curva inicial de fluorescencia Despliegue <ol><li> Una solución stock de 10 M de urea en 20 mM tampón fosfato (pH 7,3) se hace. Es esencial que el agua se añade a la sólida (y no al revés), ya que la gran cantidad de urea ocupa un volumen importante en la solución. Esta solución de urea se puede hacer mediante la adición de solutos a una botella limpia, vacía y añadir agua hasta el volumen final previsto. Esta solución puede requerir un calentamiento en un baño de agua o sobre una plancha caliente con agitación para disolver el soluto. La urea es higroscópica y por lo tanto, la concentración real de la solución de urea debe ser determinada mediante el índice de refracción 1. </li><li> Una solución de reserva de estabilización de fosfato 20 mM (pH 7,3) se hace. </li><li> Una solución de reserva de proteínas desplegadas se prepara. Esta solución de proteínas de reserva debe tener una concentración de proteína M ~ 200 en urea 8 M, 20 mM tampón fosfato. </li><li> Las muestras para los estudios de fluorescencia se preparan de la siguiente manera. Los volúmenes adecuados de proteína de valores (sección 2.3), solución de reserva de lípidos (5 mg / mL de lípidos, la sección 1), las acciones de solución de urea 10 M (sección 2.1), y la Reserva de Estabilización de fosfato (sección 2.2) se combinaron para hacer de las muestras que contienen ~ 4 proteína M, 1 mg / mL de lípidos, y de 0 a 8 M urea en incrementos de 1 M. El volumen total de cada muestra es de 200 mL. Una hoja de ejemplo (tabla 1) se incluye que los volúmenes de listas requiere el uso de 200 acciones M solución de proteína. </li><li> Muestras en blanco se hacen como se describe en la sección 2.4, sin embargo, la proteína no se añade. En lugar de proteínas, 4 l de una solución de urea 8 M se puede añadir que las concentraciones de otros componentes son idénticos a los de la sección 2.4. Estos espacios en blanco se utiliza para restar la dispersión y la señal de fondo otra que aparece en el espectro de fluorescencia de la proteína. </li><li> Las muestras de las secciones 2.4 y 2.5 se permite que se incuba a 37 ° C durante al menos 2 horas antes de la medición de los espectros de fluorescencia para asegurar plegado completo y equilibrado. </li></ol> 3. Medición de los espectros de fluorescencia Fluorescencia de triptófano de cada muestra y el blanco se mide utilizando un fluorómetro de estado estacionario. Espectros de fluorescencia debe registrarse con longitud de onda de excitación de 290 nm para evitar la excitación de los residuos de tirosina, y escaneado 305 a 500 nm. Usual de ingreso y paso de banda de salida es de 3 nm. El incremento de longitud de onda y el tiempo de integración puede ser optimizado para la relación señal-ruido. Los valores típicos para el incremento de longitud de onda y el tiempo de integración 1 nm / paso y 0.5 seg / paso, respectivamente. Proteínas de la membrana en general, se pliegan en lípidos sintéticos sólo cuando la temperatura está por encima de la temperatura de la fase de transición de dos capas. Por lo tanto, en este caso de DMPC, la temperatura de la muestra se mantiene constante a 30 º C. Una cubeta microvolumen sílice fundido con capacidad de 160 l se utiliza en estos exexperimentos. 4. Generación de la curva inicial de despliegue y la estimación de la energía libre de Gibbs de desplegamiento de proteínas de membrana <ol><li> Espectros de fluorescencia en la forma de un conjunto de datos xy se cargan en el software como Igor Pro, Matlab, origen, o Excel. </li><li> Señal desde el fondo de la vesícula urea y de los lípidos se debe restar mediante el siguiente procedimiento: Espectro de A = espectro de fluorescencia de la proteína cruda en la presencia de vesículas de lípidos y la concentración específica de la urea. Espectro B = espectro de fluorescencia de crudo de la muestra en blanco correspondiente que contiene vesículas de lípidos y la concentración de urea mismo que en el espectro de A, no es la proteína en las muestras en blanco. Espectro corregido = Espectro A - C * espectro B. C es un escalar por lo general igual a 1,0. En algunos casos, C es inferior o superior a 1,0 debido a problemas de dispersión que causa sobre o sub-resta cerca de la región 305-320 nm ~. </li><li> La longitud de onda del máximo de fluorescencia, λ max, está tabulada de los espectros corregidos para cada concentración de urea. Un valor típico para desplegada completamente proteína de la membrana es de 350 nm, mientras que la proteína plegada es de ~ 330 nm. Longitudes de onda tabulados se convierten en la fracción se desarrolló mediante la conversión de la gama de valores λ max (~ 330 a ~ 350 nm) para el rango de 0,0 a 1,0 para correlacionar el valor de λ max a la fracción de la población se desarrolló. Por ejemplo, un valor máximo de λ de 330 nm corresponde a 0,0, 350 nm corresponde a 1,0 y 340 nm corresponde a 0,5 en términos de la fracción se desarrolló. Esta fracción de la proteína desplegada se traza contra la concentración de urea. Como se mencionó anteriormente, la concentración de urea se debe determinar experimentalmente midiendo el índice de refracción. </li><li> Suponemos que la proteína puede existir en dos estados: plegar o desplegar. La trama de la fracción se desarrolló, f, frente a la concentración de urea, C, puede estar en forma para la ecuación: 2 <img src="/files/ftp_upload/2669/2669eq2.jpg" alt="La ecuación 2" /> El coeficiente m corresponde a la tasa de cambio de la energía libre con respecto a la concentración de desnaturalizante, y C m corresponde a la concentración de urea en el punto medio en el que la población es igual a doblado la población se desarrolló. Estos valores son las variables de ajuste obtenidos a partir de los mínimos cuadrados instalación de software de análisis de datos. R (0.001987 kcal / mol • K) es la ley constante de los gases, y T es la temperatura (303 K). </li><li> Los coeficientes obtenidos del ajuste se utilizan para determinar la energía libre de Gibbs (kcal / mol) de desarrollo en ausencia de urea: <img src="/files/ftp_upload/2669/2669eq3.jpg" alt="La ecuación 3"/> . </li></ol> 5. Despliegue optimizado para la curva de medición más precisa de la energía libre de Gibbs de despliegue. <ol><li> La curva de desarrollo descritas anteriormente revela el rango de concentraciones de urea que provoca el mayor cambio en el máximo de λ. Este rango es generalmente pequeña, y la mayoría de despliegue se produce dentro de este rango pequeño. Con el fin de determinar con precisión la energía libre de desarrollo, se analizan muestras adicionales en esta región para obtener una gráfica que contiene muchos puntos de datos necesarios para la optimización de mínimos cuadrados de montaje. Por ejemplo, si la proteína se desarrolla entre 2 y 4 M de urea, las muestras se puede decir que contiene 2 a 4 M de urea en incrementos de 0,2 M a revelar con mayor precisión la forma de esta importante región de la curva de desarrollo. No es necesario medir el espectro en blanco en cada concentración de urea, sino que es suficiente para medir un espectro blanco cada paso ~ 0,5 M. </li><li> La energía libre de desarrollo obtenidos a partir de esta trama puede diferir ligeramente de los obtenidos en la curva original desarrollo, sino que refleja un valor más preciso. </li></ol> 6. Los resultados representativos: <table border="1"><tbody><tr><td> Muestra </td><td> final [urea], M </td><td> proteína de acciones (l) </td><td> de valores de lípidos (l) </td><td> stock 10 M urea (l) </td><td> reserva de estabilización (l) </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> Tabla 1. Volumen de solución madre necesaria para hacer muestras de fluorescencia <img src="/files/ftp_upload/2669/2669fig1.jpg" alt="Figura 1" /> Figura 1. Triptófano espectros de fluorescencia de la proteína de ~ 5 M de membrana representante que contiene un único residuo de triptófano. (A) de los espectros de fluorescencia de la proteína cruda (espectro A partir de 4.2), (B) de los espectros de fluorescencia de crudo en blanco (espectro B de 4,2), (C) Se ha corregido espectro de 4,2. <img src="/files/ftp_upload/2669/2669fig2.jpg" alt="Figura 2" /> Figura 2. Corregido triptófano espectros de fluorescencia de la proteína de la membrana de la Figura 1 para las concentraciones de urea numerosas. <img src="/files/ftp_upload/2669/2669fig3.jpg" alt="Figura 3" /> Figura 3. Despliegue curva obtenida a partir de datos en la figura 2, con ajuste a la ecuación en el 4,4. La energía libre de Gibbs se calcula de acuerdo con la sección 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>

El actual protocolo describe la generación de curvas de desarrollo asociados a la membrana de las proteínas y los péptidos que contienen residuos de triptófano. En este caso, se asume que la fluorescencia de triptófano refleja si la proteína se dobla y se introduce en las vesículas de lípidos sintéticos, o se desarrolló en la solución. Supuestos adicionales, como el plegado de dos estados y la dependencia lineal de energía libre con la concentración de desnaturalizante, se hacen en el presente informe;. Mod...

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<td>DMPC</td>
<td>&nbsp;</td>
<td>Avanti Polar Lipids</td>
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thermodynamics of membrane protein folding measured by fluorescence spectroscopy

Plegamiento de proteínas de membrana es un tema emergente con un significado fundamental y relacionadas con la salud. La abundancia de proteínas de membrana en las células subyace la necesidad de un estudio integral del plegamiento de esta familia de proteínas en todas partes. Además, los avances en nuestra capacidad para caracterizar las enfermedades asociadas con las proteínas mal plegadas, han motivado importantes esfuerzos experimentales y teóricos en el campo de plegamiento de proteínas. Los rápidos avances en este campo tan importante es, por desgracia obstaculizado por los problemas inherentes asociados con las proteínas de membrana y de la complejidad del mecanismo de plegado. Aquí, se describe un procedimiento experimental para la medición de la propiedad termodinámica de la energía libre de Gibbs se desarrolla en ausencia de desnaturalizante, Δ G ° H 2 O, una proteína de membrana integral representante de E. coli. Este protocolo se centra en la aplicación de la espectroscopia de fluorescencia para determinar las poblaciones de equilibrio de los estados plegado y desplegado en función de la concentración de desnaturalizante. Consideraciones experimentales para la preparación de vesículas de lípidos sintéticos, así como pasos clave en el procedimiento de análisis de datos se destacan. Esta técnica es muy versátil y puede llevarse a cabo con diferentes tipos de desnaturalizante, incluyendo la temperatura y pH, así como en diversos entornos de plegado de los lípidos y las micelas. El protocolo actual es el que puede ser generalizado a cualquier membrana o proteínas solubles que se reúne el conjunto de los criterios expuestos.

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 artículo detalla el procedimiento de vídeo experimental para la obtención de la energía libre de Gibbs del plegamiento de las proteínas de membrana de triptófano fluorescencia.

Termodinámica del plegado de proteínas de membrana medido por espectroscopia de fluorescencia

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 vistas.  University of California San Diego - UCSD. Este artículo detalla el procedimiento de vídeo experimental para la obtención de la energía libre de Gibbs del plegamiento de las proteínas de membrana de triptófano fluorescencia.

Video: 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 detección de fluorescencia para las plataformas de las gotas de microfluidos

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

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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).

Mezcladores de microfluidos para el estudio plegamiento 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-COLUMNA: una herramienta bioquímica para identificar las interacciones proteína-proteína de Proteínas de la Membrana<em&gt; En 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 resolución espacio-temporal Análisis de la Dinámica del receptor por Casa molécula de microscopía de fluorescencia

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.

Rastreo Orbital 3D en un microscopio de dos fotones Modificado: una aplicación para el Seguimiento 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.

Preparación de la 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).

Ingeniería 'Oro' de la fluorescencia por presión selectiva incorporación de aminoácidos no canónica y análisis de proteínas por espectrometría de masas y la fluorescencia

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.

Detección de endotoxinas en las formulaciones Nano mediante ensayos de Limulus Amoebocyte Lysate (LAL)

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.

En Vesiculo Síntesis de Precursores de Membrana De péptido para crecimiento de la vesícula Autónoma

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.

Análisis constante de resorte molecular mediante espectroscopia de sonda de fuerza de 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 ...