Name: use of microscale thermophoresis to measure protein-lipid interactions
Uploaded: 2022-02-10T12:30:00
Description: Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

Esta investigación fue apoyada por una subvención de la National Science Foundation (MCB 1818310) a la RAF. Este trabajo fue apoyado en parte por la Instalación Básica de Biología Química / Unidad de Cristalografía de Proteínas en el Centro oncológico H. Lee Moffitt (NIH / NCI: P30-CA076292).

1. Preparación de materiales
<ol><li>Preparar solución salina tamponada con fosfato (PBS): 137 mM NaCl, 2.5 mM KCl, 10 mM NaH2PO4 y 2 mM KH2PO4, pH 7.41.</li>
	<li>Preparar el tinte NTA-Atto 647 N. Diluya el tinte NTA-Atto 647 N a 100 nM de una solución 100% DMSO en PBS sin Tween.</li>
	<li>Express Vam7-His8 – Proteína como proteína de fusión en E. coli y purificar mediante Cromatografía de Ni-NTA y exclusión de tam...

<ol>
	<li>Jerabek-Willemsen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroScale+Thermophoresis:+Interaction+analysis+and+beyond.">MicroScale Thermophoresis: Interaction analysis and beyond.</a> Journal of Molecular Structure. 1077, 101-113 (2014).</li><li>Chapman, D., Hochstrasser, R., Millar, D., Youderia, n. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+proteins+without+primary+sequence+tryptophan+residues:+mutant+trp+repressors+with+aliphatic+substitutions+for+tryptophan+side+chains.">Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains.</a> Gene. 163 (1), 1-11 (1995).</li><li>Duff, M. R., Grubbs, J., Howell, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal+titration+calorimetry+for+measuring+macromolecule-ligand+affinity.">Isothermal titration calorimetry for measuring macromolecule-ligand affinity.</a> Journal of Visualized Experiments. (55), (2011).</li><li>Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interaction+studies+using+microscale+thermophoresis.">Molecular interaction studies using microscale thermophoresis.</a> Assay and Drug Development Technologies. 9 (4), 342-353 (2011).</li><li>Wienken, C. J., Baaske, P., Rothbauer, U., Braun, D., Duhr, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-binding+assays+in+biological+liquids+using+microscale+thermophoresis.">Protein-binding assays in biological liquids using microscale thermophoresis.</a> Nature Communications. 1, 100 (2010).</li><li>Khavrutskii, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+purification-free+method+of+binding+affinity+determination+by+microscale+thermophoresis.">Protein purification-free method of binding affinity determination by microscale thermophoresis.</a> Journal of Visualized Experiments. (78), (2013).</li><li>Seidel, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-free+microscale+thermophoresis+discriminates+sites+and+affinity+of+protein-ligand+binding.">Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.</a> Angewandte Chemie International Edition English. 51 (42), 10656-10659 (2012).</li><li>Starr, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidic+acid+induces+conformational+changes+in+Sec18+protomers+that+prevent+SNARE+priming.">Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.</a> Journal of Biological Chemistry. 294 (9), 3100-3116 (2019).</li><li>Miner, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Central+Polybasic+Region+of+the+Soluble+SNARE+(Soluble+N-Ethylmaleimide-sensitive+Factor+Attachment+Protein+Receptor)+Vam7+Affects+Binding+to+Phosphatidylinositol+3-Phosphate+by+the+PX+(Phox+Homology)+Domain.">The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.</a> Journal of Biological Chemistry. 291 (34), 17651-17663 (2016).</li><li>Collins, M. D., Gordon, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+phosphoinositide+partitioning+into+plasma+membrane+models.">Short-chain phosphoinositide partitioning into plasma membrane models.</a> Biophysical Journal. 105 (11), 2485-2494 (2013).</li><li>Zhao, H., Lappalainen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+guide+to+biochemical+approaches+for+analyzing+protein-lipid+interactions.">A simple guide to biochemical approaches for analyzing protein-lipid interactions.</a> Molecular Biology of the Cell. 23 (15), 2823-2830 (2012).</li><li>Kaufmann, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+proteins:+virulence+factors+of+intracellular+disease+agents.">Stress proteins: virulence factors of intracellular disease agents.</a> Immunitat und Infektion. 17 (4), 124-128 (1989).</li><li>le Maire, M., Champeil, P., Moller, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+membrane+proteins+and+lipids+with+solubilizing+detergents.">Interaction of membrane proteins and lipids with solubilizing detergents.</a> Biochimica et Biophysica Acta. 1508 (1-2), 86-111 (2000).</li><li>Corradi, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+Diversity+in+Lipid-Protein+Interactions.">Emerging Diversity in Lipid-Protein Interactions.</a> Chemical Reviews. 119 (9), 5775-5848 (2019).</li><li>Rainard, J. M., Pandarakalam, G. C., McElroy, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Microscale+Thermophoresis+to+Characterize+Hits+from+High-Throughput+Screening:+A+European+Lead+Factory+Perspective.">Using Microscale Thermophoresis to Characterize Hits from High-Throughput Screening: A European Lead Factory Perspective.</a> SLAS Discovery. 23 (3), 225-241 (2018).</li><li>Sparks, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small-molecule+competitive+inhibitor+of+phosphatidic+acid+binding+by+the+AAA++protein+NSF/Sec18+blocks+the+SNARE-priming+stage+of+vacuole+fusion.">A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.</a> Journal of Biological Chemistry. 294 (46), 17168-17185 (2019).</li></ol>

La determinación de Vam7-His8unión a DiC8-PA proporcionó un KD ajustado robusto para la interacción dada, que es una afinidad ligeramente menor que el KD medido de Vam7-His8 a los liposomas pa (no publicado). Esta diferencia se debe muy probablemente a la falta de una membrana, lo que generalmente resulta en una menor afinidad por las interacciones de unión lipídica específicas de la membrana y, por lo tanto, demuestra el papel del andamio de membrana del l...

La termoforesis a microescala es una técnica relativamente nueva para determinar constantes de disociación (KD) y constantes de inhibición (IC50) entre ligandos bioquímicamente relevantes. El minorista comercial líder para MST (por ejemplo, NanoTemper) ofrece dos tecnologías populares de MST: 1) MST sin etiquetas que requiere una etiqueta fluorescente, y 2) termoforesis etiquetada utilizando la fluorescencia inherente de proteínas dependientes del número de residuos aromáticos presentes en cada proteína1. Una desventaja de la termoforesis sin etiqueta es que, en la mayoría de los casos, no permite la medición de las interacciones proteína-proteína. Sin embargo, puede ser posible diseñar proteínas sin aminoácidos aromáticos como el triptófano para su uso en la termoforesis sin etiqueta2.
MST mide el movimiento de partículas en respuesta a la inducción de campos de temperatura microscópicos iniciados por un láser infrarrojo en las tecnologías actualmente disponibles1. MST se puede utilizar para medir las interacciones proteína-proteína, las interacciones proteína-lípido, la molécula pequeña de proteína, los experimentos de competencia e incluso las interacciones entre moléculas pequeñas, siempre y cuando se pueda producir suficiente separación de señales. Además, MST permite la medición de interacciones basadas en membrana-proteína incrustadas en liposomas o nanodiscos. La termoforesis etiquetada aprovecha el uso de etiquetas marcadas fluorescentemente que permiten la separación químicamente controlable de la señal entre el ligando y el analito. Los valores de KD se pueden obtener mediante termoforesis para interacciones que implican la unión a proteínas a bajas concentraciones nanomolares, que en la mayoría de los casos es una concentración de proteína mucho menor que la requerida para la calorimetría isotérmica (ITC)3. Además, MST no tiene requisitos de amortiguación estrictos como se requiere para la resonancia de plasmón de superficie (SPR)4 y la termoforesis etiquetada incluso se puede usar para medir constantes de unión de proteínas de interés a partir de soluciones proteicas no completamente purificadas5 con etiquetas fluorescentes insertadas genéticamente6. Una desventaja de MST es que los parámetros cinéticos no se pueden obtener fácilmente para MST como en SPR2.
Las mediciones de termoforesis dependen de la diferencia de temperatura local de una solución. Este calor se puede generar a partir de un láser infrarrojo. El dispositivo MST tiene un detector de fluorescencia acoplado a un haz infrarrojo (IR) y puede detectar cambios en la fluorescencia de los cambios de concentración local de las moléculas fluorescentes en el punto donde se dirige el láser IR. El dispositivo MST utiliza un láser dirigido a IR acoplado directamente a un detector de fluorescencia enfocado en el mismo punto en el que se genera el calor en la solución. Esto permite una detección robusta de los cambios de temperatura correspondientes al agotamiento de las moléculas en el punto de calor generado por el láser IR. La fluorescencia medida generalmente disminuye más cerca del láser IR en respuesta a los aumentos de temperatura. Las diferencias medidas como resultado pueden deberse a múltiples factores, incluida la carga, el tamaño o la entropía de la solvatación. Estas diferencias se miden como cambios en la fluorescencia en respuesta a la inducción de calor o al movimiento de moléculas de partes calientes a más frías del capilar.
Al cargar un capilar con una solución dada, es importante dejar aire en cada extremo del capilar y no cargar el capilar completamente lleno. El capilar comercial contiene aproximadamente 10 μL de solución. Uno puede lograr mediciones precisas con 5 μL de solución siempre que la solución se manipule hasta el centro del capilar, no haya burbujas de aire (potencialmente desgasificación antes de la carga capilar), y uno tenga cuidado al cargar el bastidor para no empujar la solución desde el centro del capilar, donde se dirige el láser infrarrojo. Si el láser no entra en contacto completamente con la solución, lo más probable es que el resultado sea una de las tres salidas inutilizables para esa concentración: 1) detección de fluorescencia nula o baja, 2) detección de fluorescencia más alta (potencialmente con pico irregular), o 3) detección de fluorescencia dentro de otros valores de titulación dada, pero con un pico irregular y no redondeado.
Para la termoforesis etiquetada es óptimo tener una señal de fluorescencia por encima de 200 y por debajo de 2000 unidades de fluorescencia7. El dispositivo MST utiliza un rango de intensidades LED de 0 a 100, que se pueden seleccionar para lograr una señal superior a 200 o inferior a 2000. Alternativamente, se pueden usar diferentes concentraciones del ligando marcado para modificar la señal de fluorescencia a un nivel óptimo. Es importante ejecutar un escaneo de tapa con una medición MST dada como referencia al analizar datos, ya que un escaneo de tapa deficiente a menudo puede resultar en un punto que luego se puede determinar que es un valor atípico. Cada carrera debe tomar aproximadamente 30 minutos si se mide una sola potencia MST con un escaneo de tapa. Los dispositivos comerciales permiten cambios en la potencia MST. En versiones de software anteriores, esto se podía establecer de 0 a 100; y en versiones posteriores se puede seleccionar MST bajo, medio o alto. Para lograr trazas robustas, un investigador puede necesitar probar cada una de ellas y decidir qué configuración de MST da como resultado los datos más robustos para una interacción determinada.

<table>
	<tbody>
		<tr>
			<td>Cy5 Maleimide Mono-Reactive Dye</td>
			<td>GE Healthcare</td>
			<td>PA23031</td>
			<td>For protein labeleing</td>
		</tr>
		<tr>
			<td>Graphpad Prsim</td>
			<td>Graphpad software</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monolith NT.115 Capillaries (1000 count)</td>
			<td>Nanotemper</td>
			<td>MO-K022</td>
			<td>Capillaries for MST</td>
		</tr>
		<tr>
			<td>Monolith NT.115 machine</td>
			<td>Nanotemper</td>
			<td></td>
			<td>University equipment</td>
		</tr>
		<tr>
			<td>NTA-Atto 647 N</td>
			<td>Sigma</td>
			<td>2175</td>
			<td>label for His tags</td>
		</tr>
		<tr>
			<td>Phosphatidylinositol 3-phosphate diC8 (PI(3)P diC8)</td>
			<td>Echelon</td>
			<td>P-3008</td>
			<td>Lipid for binding experiments</td>
		</tr>
	</tbody>
</table>

use of microscale thermophoresis to measure protein-lipid interactions

La capacidad de determinar la afinidad de unión de los lípidos a las proteínas es una parte esencial de la comprensión de las interacciones proteína-lípido en el tráfico de membranas, la transducción de señales y la remodelación citoesquelética. Las herramientas clásicas para medir tales interacciones incluyen la resonancia de plasmón de superficie (SPR) y la calorimetría de titulación isotérmica (ITC). Si bien son herramientas poderosas, estos enfoques tienen contratiempos. ItC requiere grandes cantidades de proteína purificada, así como lípidos, que pueden ser costosos y difíciles de producir. Además, el ITC y el SPR consumen mucho tiempo, lo que podría aumentar significativamente el costo de realizar estos experimentos. Una forma de evitar estas restricciones es utilizar la técnica relativamente nueva de termoforesis a microescala (MST). MST es rápido y rentable utilizando pequeñas cantidades de muestra para obtener una curva de saturación para un evento de unión dado. Actualmente hay dos tipos de sistemas MST disponibles. Un tipo de MST requiere etiquetado con un fluoróforo en el espectro azul o rojo. El segundo sistema se basa en la fluorescencia intrínseca de los aminoácidos aromáticos en el rango UV. Ambos sistemas detectan el movimiento de las moléculas en respuesta a la inducción localizada de calor de un láser infrarrojo. Cada enfoque tiene sus ventajas y desventajas. El MST sin etiquetas puede usar proteínas nativas sin etiquetar; sin embargo, muchos analitos, incluidos los productos farmacéuticos, emiten fluorescencia en el rango UV, lo que puede interferir con la determinación de valores precisos de KD. En comparación, el MST etiquetado permite una mayor diversidad de interacciones medibles por pares utilizando sondas marcadas fluorescentemente unidas a ligandos con absorbancias medibles en el rango visible en lugar de UV, lo que limita el potencial de interferencia de señales de analitos.

Esta es una salida de muestra utilizando el análisis de afinidad. El MST etiquetado se utilizó para determinar la constante de unión del Vam7-His8 al dioctanoilo soluble (DiC8) PA de uno de sus sustratos naturales9. La Figura 1 presenta los rastros termoforéticos de un ensayo de una titulación 1:1 de DiC8 PA a partir de 500 μM contra 50 nM de Vam7-His8. Se muestra la fluorescencia inicial (tiempo antes de que se encienda el láser infrarrojo), Tjump (tiempo inic...

Watch this Scientific Journal Video about Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions at JoVE.com

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

La termoforesis a microescala obtiene constantes de unión rápidamente a bajo costo de material. La termoforesis a microescala etiquetada o sin etiqueta está disponible comercialmente; sin embargo, la termoforesis sin etiquetas no es capaz de la diversidad de mediciones de interacción que se pueden realizar utilizando etiquetas fluorescentes. Proporcionamos un protocolo para mediciones de termoforesis etiquetadas.

Uso de la termoforesis a microescala para medir las interacciones proteína-lípido

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

La termoforesis a microescala etiquetada obtiene constantes de disociación, o KD, de diversas interacciones bioquímicas de manera eficiente. Este protocolo y el vídeo que lo acompaña dan la afinidad aproximada por Vam7, la única proteína SNARE soluble en levadura, al ácido fosfatídico. Esta es la forma en que Vam7 se asocia con dos membranas antes de interactuar con otras proteínas.Este método obtiene KDs rápidamente para diversas interacciones bioquímicas a bajo costo, con alta reproducibilidad y con un costo mínimo de proteínas. Esta técnica es adecuada para el desarrollo farmacéutico en primera etapa, lo que permite que los KD fácilmente obtenibles determinen posibles compuestos de plomo. Antes de comenzar un análisis, encienda el interruptor de encendido en la parte posterior del dispositivo MST y abra el software de control.Confirme que el equipo está conectado al dispositivo y en estado conectado y establezca el MST Antes en 3 segundos, el MST en 30 segundos y la Recuperación de fluorescencia en después de 1 segundo. Para cada tubo capilar, ingrese el nombre del ligando objetivo, el nombre del analito del ligando, la concentración del objetivo y la concentración de titulación más alta utilizando la relación de titulación de autollen. Seleccione un rango de potencias MST e introduzca valores para cada potencia para probar el ajuste de unión más robusto.Para preparar muestras de MST etiquetadas, diluya una solución de Vam7-octahistidina a una concentración de 200 nanomolares y un colorante NTA-Atto 647 a una concentración nanomolar de 100, ambos en PBS. Mezcle el Vam7-octahistidina y el tinte NTA-Atto 647 en una proporción volumétrica de 1:1 y deje que la mezcla permanezca a temperatura ambiente protegida de la luz durante 30 minutos. Al final de la incubación, centrífuga la mezcla de colorante y proteína y almacene la solución a 4 grados centígrados hasta unas horas antes de su uso.Luego, prepare la muestra, tome el ligando teñido y exponga el analito. Para la termoforesis a microescala de la muestra, abra el dispositivo y deslice el estante capilar hacia afuera. Cargue los capilares de muestra en el bastidor con la mayor concentración en la posición uno y seleccione el canal rojo en el software de control.Luego, cargue el bastidor en el instrumento. A continuación, seleccione un rango de potencia MST e inicie la medición MST de Cap Scan y evalúe la respuesta de desplazamiento. Aquí se muestran los rastros termoforéticos de un ensayo de una titulación 1:1 de DiC8 PA a partir de 500 micromoles contra 50 nanomolares NTA-Atto 647 marcados como dominio Vam7.Se puede observar la fluorescencia inicial, el salto de tiempo y la termoforesis, lo que permite calcular KD a partir de cualquiera o una combinación de estas mediciones. Se puede trazar una curva de saturación a partir de estos resultados, por ejemplo, para la termoforesis con la salida de salto de tiempo, como se ilustra en el gráfico. Generalmente, los resultados de MST se determinan utilizando la escala logarítmica, teniendo en cuenta la concentración de proteínas para determinar la afinidad de unión seleccionando el modelo KD e ingresando la concentración de proteínas.Al realizar este protocolo, asegúrese de que la solución esté en el centro del capilar y que no haya burbujas de aire y que el capilar no tenga polvo o solución en el exterior del capilar. La calorimetría de titulación isotérmica o la resonancia de plasmón de superficie podrían utilizarse para validar el KD experimental obtenido. Si existe una posible vinculación cooperativa, el ITC sería el siguiente método lógico realizado, dados los recursos.Esta técnica ha permitido a los investigadores de biología tradicional incorporar técnicas de detección biofísica en su investigación para determinar las relaciones de vía. Un ejemplo de esta técnica es la lisasa celular con la proteína diana expresada endógenamente, donde la etiqueta GFP es una forma novedosa de realizar un ensayo tradicional de pull-down.

use-of-microscale-thermophoresis-to-measure-protein-lipid-interactions

Research

JoVE Journal

Biochemistry

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

Mapear el sitio de unión de un aptámero de ATP utilizando MicroScale termoforesis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Investigación

Bioquímica

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

La inmunofluorescencia cuantitativa a medida Global localizada traducción

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

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.

Péptidos de penetración celular biotinilado para estudiar interacciones de proteínas intracelulares

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

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin repeats. Here, a protocol for detecting and measuring such interactions is described using the globular plakin repeat domain of envoplakin and the helical coil of vimentin. This provides a basis for determining whether a protein binds vimentin (or similar filamentous proteins) and for measurement of the affinity of the interaction. The globular protein of interest is labeled with 15N and titrated with vimentin protein in solution. A two-dimensional NMR spectrum is acquired to detect interactions by observing changes in peak shape or chemical shifts, and to elucidate effects of solution conditions including salt levels, which influence vimentin quaternary structure. If the protein of interest binds the filamentous ligand, the binding interaction is quantified by MST using the purified proteins. The approach is a straightforward way for determining whether a protein of interest binds a filament, and for assessing how alterations, such as mutations or solution conditions, affect the interaction.

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy NMR and Microscale Thermophoresis MST

Here, we present a protocol for the production and purification of proteins that are labeled with stable isotopes, and subsequent characterization of protein-protein interactions using Nuclear Magnetic Resonance (NMR) spectroscopy and MicroScale Thermophoresis (MST) experiments.

Medición de las interacciones de las proteínas globulares y filamentosas por espectroscopia de resonancia magnética Nuclear (RMN) y microescala Thermophoresis (MST)

Filamentous proteins such as vimentin provide organization within cells by providing a structural scaffold with sites that bind proteins containing plakin ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in proteins and generating redox stress. Pathological conditions that lead to metal depletion or accumulation are causal agents of different human diseases. Some examples include anemia, acrodermatitis enteropathica, and Wilson&#8217;s and Menkes&#8217; diseases. It is therefore important to be able to measure the levels and transport of transition metals in biological samples with high sensitivity and accuracy in order to facilitate research exploring how these elements contribute to normal physiological functions and toxicity. Zinc (Zn), for example, is a cofactor in many mammalian proteins, participates in signaling events, and is a secondary messenger in cells. In excess, Zn is toxic and can inhibit absorption of other metals, while in deficit, it can lead to a variety of potentially lethal conditions.

Graphite furnace atomic absorption spectroscopy (GF-AAS) provides a highly sensitive and effective method for determining Zn and other transition metal concentrations in diverse biological samples. Electrothermal atomization via GF-AAS quantifies metals by atomizing small volumes of samples for subsequent selective absorption analysis using wavelength of excitation of the element of interest. Within the limits of linearity of the Beer-Lambert Law, the absorbance of light by the metal is directly proportional to concentration of the analyte. Compared to other methods of determining Zn content, GF-AAS detects both free and complexed Zn in proteins and possibly in small intracellular molecules with high sensitivity in small sample volumes. Moreover, GF-AAS is also more readily accessible than inductively coupled plasma mass spectrometry (ICP-MS) or synchrotron-based X-ray fluorescence. In this method, the systematic sample preparation of different cultured cell lines for analyses in a GF-AAS is described. Variations in this trace element were compared in both whole cell lysates and subcellular fractions of proliferating and differentiated cells as proof of principle.

Cultured primary or established cell lines are commonly used to address fundamental biological and mechanistic questions as an initial approach before using animal models. This protocol describes how to prepare whole cell extracts and subcellular fractions for studies of zinc (Zn) and other trace elements with atomic absorbance spectroscopy.

Espectroscopía de absorbancia atómica para medir las agrupaciones intracelulares de zinc en células de mamífero

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Uso de Minigenes que contienen elementos Alu para analizar ARN circulares

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of PNKs can be quantified using direct or indirect approaches. Presented here is a direct, in vitro approach to measure PNK activity that relies on a fluorescently-labeled oligonucleotide substrate and polyacrylamide gel electrophoresis. This approach provides resolution of the phosphorylated products while avoiding the use of radiolabeled substrates. The protocol details how to set up the phosphorylation reaction, prepare and run large polyacrylamide gels, and quantify the reaction products. The most technically challenging part of this assay is pouring and running the large polyacrylamide gels; thus, important details to overcome common difficulties are provided. This protocol was optimized for Grc3, a PNK that assembles into an obligate pre-ribosomal RNA processing complex with its binding partner, the Las1 nuclease. However, this protocol can be adapted to measure the activity of other PNK enzymes. Moreover, this assay can also be modified to determine the effects of different components of the reaction, such as the nucleoside triphosphate, metal ions, and oligonucleotides.

This protocol describes a nonradioactive assay to measure kinase activity of polynucleotide kinases (PNKs) on small DNA and RNA substrates.

Ensayo no radioactivo para medir la fosforilación de polinucleótido de sustratos de nucleótidos pequeños

Polynucleotide kinases (PNKs) are enzymes that catalyze the phosphorylation of the 5&#39; hydroxyl end of DNA and RNA oligonucleotides. The activity of ...

CD Spectroscopy to Study DNA-Protein Interactions

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent advancements in CD spectroscopy have enabled the study of DNA-protein interactions and conformational dynamics of DNA in different microenvironments in detail for a better understanding of transcriptional regulation in vivo. The area around a potential transcription zone needs to be unwound for transcription to occur. This is a complex process requiring the coordination of histone modifications, binding of the transcription factor to DNA, and other chromatin remodeling activities. Using CD spectroscopy, it is possible to study conformational changes in the promoter region caused by regulatory proteins, such as ATP-dependent chromatin remodelers, to promote transcription. The conformational changes occurring in the protein can also be monitored. In addition, queries regarding the affinity of the protein towards its target DNA and sequence specificity can be addressed by incorporating mutations in the target DNA. In short, the unique understanding of this sensitive and inexpensive method can predict changes in chromatin dynamics, thereby improving the understanding of transcriptional regulation.

The interaction of an ATP-dependent chromatin remodeler with a DNA ligand is described using CD spectroscopy. The induced conformational changes on a gene promoter analyzed by the peaks generated can be used to understand the mechanism of transcriptional regulation.

Espectroscopia de CD para estudiar las interacciones ADN-proteína

Circular dichroism (CD) spectroscopy is a simple and convenient method to investigate the secondary structure and interactions of biomolecules. Recent ...

Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates.&#160;As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

This method describes a lectin-based in vitro sedimentation assay to quantify the binding affinity of glucan phosphatase and amylopectin. This co-sedimentation assay is reliable for measuring glucan phosphatase substrate binding and can be applied to various solubilized glucan substrates.

Ensayo de sedimentación basado en concanavalina A para medir la unión al sustrato de las fosfatasas de glucano

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals ...

Rastreo de isótopos estables y análisis de LC-MS para medir las actividades de DHODH y SDH

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

Autor destacado: Ensayos independientes del oxígeno para medir la función mitocondrial en mamíferos  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

Ensayos independientes del oxígeno para medir la función mitocondrial en mamíferos

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...