VJ y HS reconocen el apoyo de NIH R01 GM094246 a VJ. HS reconoce fondos de puesta en marcha del Programa de Investigación Creativa de la Universidad de Clemson y el Centro de Ciencia de Materiales Ópticos y Tecnologías de Ingeniería en la Universidad de Clemson. Este proyecto también fue apoyado por una beca de formación del Centro Keck para el Entrenamiento Interdisciplinario en Biociencias de los Consorcios de la Costa del Golfo (NIGMS Subvención No. 1 T32GM089657-05) y la Beca de la Fundación Schissler para Estudios Traslacionales de Enfermedades Humanas Comunes a DD. El contenido es exclusivamente responsabilidad de los autores y no representa necesariamente las opiniones oficiales de los Institutos Nacionales de Salud.

Todos los autores declaran que no tienen intereses financieros en competencia con el contenido de este artículo.

En este trabajo se presenta el protocolo para alinear, calibrar y medir las distancias de interdye con alta precisión usando experimentos FRET de una sola molécula PIE-MFD. Al calibrar cuidadosamente todos los parámetros instrumentales, se puede aumentar la precisión de las distancias medidas y alcanzar la precisión de Angstrom. Para ello, se utilizan varios histogramas multidimensionales para analizar e identificar poblaciones para su posterior caracterización. Utilizando el tiempo macro medio para verificar la e...

Un objetivo fundamental de los estudios de biología estructural es desentrañar la relación entre la estructura y la función de las máquinas biomoleculares. La primera impresión visual de las biomoléculas ( por ejemplo, proteínas y ácidos nucleicos) se produjo en la década de 1950 a través del desarrollo de la cristalografía de rayos X 1 , 2 . La cristalografía de rayos X proporciona información estructural estática de alta resolución restringida por el empaque de cristal. Por lo tanto, la inmovilidad inherente de los modelos estructurales de rayos X evita la naturaleza dinámica de las biomoléculas, un factor que afecta a la mayoría de las funciones biológicas [ 3 , 4 , 5] . La resonancia magnética nuclear (RMN) 6 , 7 , 8 proporcionó una solución alternativa al problema resolviendo modelos estructurales en soluciones acuosas. Una gran ventajaDe la RMN es su capacidad para recuperar la naturaleza intrínseca dinámica de las biomoléculas y conjuntos conformacionales, lo que ayuda a aclarar las relaciones intrínsecas entre la estructura, la dinámica y la función 3 , 4 , 5 . Sin embargo, la RMN, limitada por el tamaño de la muestra y grandes cantidades de muestra, requiere complejas estrategias de etiquetado para sistemas más grandes. Por lo tanto, existe una necesidad acuciante de desarrollar métodos alternativos en biología estructural. Históricamente, la transferencia de energía de resonancia de Förster (FRET) 9 no ha tenido un papel importante en la biología estructural debido a la idea errónea de que FRET proporciona medidas de distancia de baja precisión. Es el propósito de este protocolo revisar la capacidad de FRET para determinar las distancias en la escala nanométrica, de tal manera que estas distancias pueden ser utilizadas para construir modelos estructurales de biomoléculas. El primer experimento verificLa dependencia de R - 6 de la eficiencia de FRET fue realizada por Stryer en 1967 10 midiendo poliprolinas de varias longitudes como una &quot;regla espectroscópica&quot;. Un experimento similar se llevó a cabo a nivel de una sola molécula en 2005 [ 11] . Las moléculas de poliprolina resultaron ser no ideales y, por lo tanto, las moléculas de ADN de doble cadena se utilizaron más tarde 12 . Esto abrió la ventana para medidas de distancia precisas y la idea de utilizar FRET para identificar las propiedades estructurales de las biomoléculas. FRET es óptimo cuando el rango de distancia entre interditos es de ~ 0.6-1.3 R 0 , donde R 0 es la distancia de Förster. Para fluoróforos típicos usados ​​en experimentos de FRET de molécula única, R $ . $ Es de aproximadamente 50 Å. Típicamente, FRET ofrece muchas ventajas sobre otros métodos en su capacidad de resolver y diferenciar las estructuras y la dinámica enHeterogéneos: i) Debido a la máxima sensibilidad de la fluorescencia, los experimentos FRET de una sola molécula 13 , 14 , 15 , 16 pueden resolver conjuntos heterogéneos contando directamente y caracterizando simultáneamente las estructuras de sus miembros individuales. (Ii) Las vías de reacción complejas pueden ser descifradas directamente en estudios FRET de molécula única porque no es necesaria la sincronización de un conjunto. (Iii) FRET puede acceder a una amplia gama de dominios temporales que abarcan más de 10 décadas en el tiempo, cubriendo una amplia variedad de dinámicas biológicamente relevantes. (Iv) Los experimentos de FRET pueden realizarse en cualquier condición de solución, tanto in vitro como in vivo . La combinación de FRET con microscopía de fluorescencia permite el estudio de estructuras moleculares y las interacciones directamente en las células vivas [ 15 , 16 , </<html> Sup&gt; 17 , 18 , 19 , incluso con alta precisión 20 . (V) FRET se puede aplicar a sistemas de casi cualquier tamaño ( por ejemplo, oligómeros de poliprolina 21 , 22 , 23 , 24 , Hsp9025, transcriptasa inversa 26 de HIV y ribosomas 27 ). (Vi) Por último, una red de distancias que contiene toda la dimensionalidad de las biomoléculas podría utilizarse para derivar modelos estructurales de moléculas estáticas o dinámicas 18 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ,Ref &quot;&gt; 35 , 36 , 37 . Por lo tanto, la espectroscopia FRET de una sola molécula se puede utilizar para derivar distancias que son lo suficientemente precisas para ser utilizado para modelado estructural restringido a distancia [ 26] . Esto es posible aprovechando la detección de fluorescencia multiparámetro 28 , 38 , 39 , 40 , 41 , 42 , que utiliza ocho dimensiones de información de fluorescencia ( es decir, espectro de excitación, espectro de fluorescencia, anisotropía, duración de la fluorescencia, rendimiento cuántico de fluorescencia, El tiempo macroscópico, las intensidades de fluorescencia y la distancia entre los fluoróforos) para proporcionar con exactitud y precisión las restricciones de distancia. Además, la excitación intercalada pulsada (PIE) se combina con MFD(PIE-MFD) 42 para monitorear la fluorescencia del aceptor de excitación directo y seleccionar eventos de molécula única que surgen de muestras que contienen una estequiometría de donante a aceptor de 1: 1. Una configuración típica de PIE-MFD usa láseres de excitación entrelazados de dos pulsos conectados a un cuerpo de microscopio confocal, donde la detección de fotones se divide en cuatro canales diferentes en diferentes ventanas espectrales y características de polarización. Más detalles se pueden encontrar en la Figura 1 . Es importante señalar que FRET debe combinarse con métodos computacionales para lograr atomístico-como los modelos estructurales que son coherentes con los resultados FRET 26 , 30 . No es el objetivo del presente protocolo revisar la metodología asociada para construir modelos estructurales con distancias derivadas de FRET. Sin embargo, estos enfoques se han aplicado en combinación con otras técnicas ( por ejemplo, dispersión de rayos X de ángulo pequeñoO la resonancia paramagnética electrónica), dando origen al campo de la biología estructural integradora 43 , 44 , 45 , 46 . El objetivo actual es allanar el camino para FRET como una herramienta cuantitativa en biología estructural. Como ejemplo, esta metodología se utilizó para identificar tres estados conformacionales en el dominio de unión al ligando (LBD) del receptor N-metil-D-aspartato (NMDA). El objetivo final es superar las limitaciones antes mencionadas y traer FRET entre los métodos integradores utilizados para la determinación estructural de biomoléculas proporcionando distancias medidas con alta precisión.

<table border="0" cellpadding="0" cellspacing="0" width="672">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">charcoal</td>
			<td style="width:183px;">Merck KGaA</td>
			<td style="width:119px;">K42964486 320</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">syringe filter</td>
			<td style="width:183px;">Fisherbrand</td>
			<td style="width:119px;">09-719C</td>
			<td>size: 0.20um</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">chambered coverglass</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td align="right" style="width:119px;">155409</td>
			<td>1.5 borosilicate glass, 8 wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">microscope cover glass</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">063014-9</td>
			<td>size: 24X60-1.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Nuclease free water</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">148859</td>
			<td>nuclease free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">tween-20</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">28320</td>
			<td>10% solution of Polysorbate 20</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (High FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">178124895</td>
			<td>5&#180;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">acceptor DNA strand (Low FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177956424</td>
			<td>5&#180;-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">donor DNA strand</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;">177951437</td>
			<td>5&#180; -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">DNA strand (No FRET)</td>
			<td style="width:183px;">Integrated DNA Technologies</td>
			<td style="width:119px;"></td>
			<td>5&#180; -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">thermal cycler</td>
			<td style="width:183px;">Eppendorf</td>
			<td style="width:119px;">E6331000025</td>
			<td>nexus gradient</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 488 C5&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A10254</td>
			<td>termed cyan-green fluorophore in the manuscript</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Alexa Fluor 647 C2&#160;Maleimide</td>
			<td style="width:183px;">Thermo Scientific</td>
			<td style="width:119px;">A20347</td>
			<td>termed far-red fluorophore in the manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Rhodamine 110</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83695-250MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Rhodamine 101</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">83694-500MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">LB Broth, Miller</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1426</td>
			<td>For culture of E. coli</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Ampicillin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:119px;">A0166</td>
			<td>Used at 100 ug/ml final concentration in selective LB medium to maintain plasmid selection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Tetracyline&#160;</td>
			<td style="width:183px;">Calbiochem</td>
			<td style="width:119px;">58346</td>
			<td>Used at 12.5 ug/ml final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Kanamycin</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP906-5</td>
			<td>Used at 15 ug/ml final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Origami B(DE3) Competent Cells</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:119px;">70837-3</td>
			<td>Competent E. coli cells for expression of protein with disulfide bridges</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Isopropyl-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:183px;">Fisher Scientific</td>
			<td style="width:119px;">BP1755</td>
			<td>For induction of E. coli protein expression</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">HiTrap Chelating HP</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td style="width:119px;">17-0409-01</td>
			<td>For Large-scale FPLC Purification of His-tagged protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Imidazole</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">56749</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Ni-NTA Agarose&#160;</td>
			<td style="width:183px;">Qiagen</td>
			<td align="right" style="width:119px;">30210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PD-10 Desalting Column</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td style="width:119px;">17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">AktaPurifier</td>
			<td style="width:183px;">GE Life Sciences</td>
			<td align="right" style="width:119px;">28406264</td>
			<td>FPLC Instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Dialysis tubing</td>
			<td style="width:183px;">Spectrum labs</td>
			<td style="width:119px;">132562</td>
			<td>15 kD MWCO 24 mm Flath width, 10 meters/roll</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Dichroics</td>
			<td style="width:183px;">Semrock</td>
			<td style="width:119px;">FF500/646-Di01-25x36</td>
			<td>500/646 BrightLight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">50/50 Beam splitter polarizer</td>
			<td style="width:183px;">Qioptiq Linos&#160;</td>
			<td style="width:119px;">G33 5743 000</td>
			<td>10x10 film polarizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Green pass filer</td>
			<td style="width:183px;">Chroma</td>
			<td style="width:119px;">ET525/50m</td>
			<td style="width:167px;">ET525/50m 25 mm diameter mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Red pass filter</td>
			<td style="width:183px;">Chroma</td>
			<td>ET720/150m</td>
			<td>ET720/150m 25 mm diameter mount</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Power Meter</td>
			<td style="width:183px;">ThorLabd</td>
			<td style="width:119px;">PM200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">UV-Vis spectrophotometer</td>
			<td style="width:183px;">Varian</td>
			<td style="width:119px;">Cary300Bio</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorolog 3 fluorometer</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">FL3-22-R3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fluorohub TCSPC controller</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">Fluorohub-B</td>
			<td>TCSPC electronics for ensemble measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 485L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">485L</td>
			<td>Blue diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">NanoLed 635L</td>
			<td style="width:183px;">Horiba</td>
			<td style="width:119px;">635L</td>
			<td>Red diode laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Olympus IX73 Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:119px;">IX73P2F</td>
			<td>Microscope frame</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 40 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932200, PMA 40</td>
			<td>Optimized for green detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">PMA 50 Hybrid Detector</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">932201, PMA 50</td>
			<td>Optimized for ed shifter sensitivity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">485nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">640nm laser</td>
			<td style="width:183px;">PicoQuant GmbH</td>
			<td style="width:119px;">LDH-D-C-640</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">Hydraharp 400 and TTTR acqusition software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">930021</td>
			<td>Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">SEPIA II SLM 828 and SEPIA software</td>
			<td style="width:183px;">PicoQuant</td>
			<td align="right" style="width:119px;">910028</td>
			<td>Laser driver for picosecond pulses , includes SEPIA software controller.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">computer</td>
			<td style="width:183px;">Dell</td>
			<td style="width:119px;">optiplex 7010</td>
			<td>cpu: i7-3770 ram:16GB</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">FRET Positioning and Screening (FPS) software</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">MFD suite</td>
			<td style="width:183px;">Heinrich Heine Unviersity</td>
			<td style="width:119px;"></td>
			<td>It includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html</td>
		</tr>
	</tbody>
</table>

high precision fret at single-molecule level for biomolecule structure determination

Aquí se presenta un protocolo sobre cómo realizar mediciones de distancia interdye de alta precisión utilizando la transferencia de energía de resonancia de Förster (FRET) a nivel de molécula única en el modo de detección de fluorescencia multiparámetro (MFD). MFD maximiza el uso de todas las &quot;dimensiones&quot; de la fluorescencia para reducir los artefactos fotofísicos y experimentales y permite la medición de la distancia interdye con una precisión de hasta 1 Å en biomoléculas rígidas. Este método se utilizó para identificar tres estados conformacionales del dominio de unión al ligando del receptor N-metil-D-aspartato (NMDA) para explicar la activación del receptor tras la unión del ligando. Al comparar las estructuras cristalográficas conocidas con mediciones experimentales, acordaron dentro de menos de 3 Å para biomoléculas más dinámicas. La reunión de un conjunto de restricciones de distancia que cubre toda la dimensionalidad de las biomoléculas haría posible proporcionar un modelo estructural de biomolécula dinámicaEs

En experimentos típicos de smFRET usando una configuración de MFD (líneas de láser: 485 nm a 60 μW y 640 nm a 23 μW, sección 5.1), la muestra de fluorescencia se diluye a una concentración picomolar baja ( 10-12 M = 1 pM) y se coloca En un microscopio confocal, donde un pulso de láser sub-nanosegundo excita las moléculas marcadas que se difunden libremente a través de un volumen de excitación. Un volumen confocal típico es &lt;4 femtolitros (fL). A concentraciones...

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High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Aquí se presenta un protocolo para experimentos FRET de alta precisión en el nivel de una sola molécula. Además, esta metodología puede usarse para identificar tres estados conformacionales en el dominio de unión al ligando del receptor de N-metil-D-aspartato (NMDA). Determinar las distancias exactas es el primer paso hacia la construcción de modelos estructurales basados ​​en experimentos FRET.

FRET de alta precisión a nivel de molécula única para la determinación de estructuras de biomoléculas

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

high-precision-fret-at-single-molecule-level-for-biomolecule

Research

JoVE Journal

Biochemistry

10.6K vistas.  Clemson University. The overall goal of this protocol is to perform high precision inter-dye distance measurements using multi-parameter fluorescence detection of F

Video: High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

La información estructural a partir de una sola molécula de FRET experimentos utilizando el Nano-posicionamiento Sistema Rápido

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

Investigación

Bioquímica

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and tissue level significantly hampers the progress in proteomic research. Results derived from currently available immunoblot techniques are also relative, preventing any efforts to combine independent studies with a large-scale analysis of protein samples. In this study, we demonstrate the process of quantitative dot blot analysis (QDB) to achieve absolute quantification in a high throughput format. Using a commercially available protein standard, we are able to determine the absolute content of capping actin protein, gelsolin-like (CAPG) in protein samples prepared from three different mouse tissues (kidney, spleen, and prostate) together with a detailed explanation of the experimental details. We propose the QDB analysis as a convenient, quantitative, high throughput immunoblot method of absolute quantification of individual proteins at the cellular and tissue level. This method will substantially aid biomarker validation and pathway verification in various areas of biological and biomedical research.

High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis QDB

Here we demonstrate a detailed process of quantitative dot blot analysis (QDB) by determining the absolute content of a targeted protein, capping actin protein, gelsolin-like (CAPG), in three different mouse tissues. We demonstrate a high throughput, convenient, quantitative immunoblot technique for biomarker validation at the cellular and tissue level.

Alto rendimiento, la absoluta determinación del contenido de una proteína seleccionada a nivel de tejido con punto cuantitativo Blot análisis (QDB)

Lacking a convenient, quantitative, high throughput immunoblot method for absolute determination of the content of a specific protein at cellular and ...

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

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

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

Uso del traste de una sola molécula a estudiar la correlación de las interacciones proteína

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

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a fluorescent dye and are spotted by a photodetector following their separation. Single molecule fluorescence imaging can provide ultrasensitive protein detection with its ability for visualizing individual fluorescent molecules. However, the application of this powerful imaging method to electrophoresis assays is hampered by the lack of ways to characterize the homogeneity of fluorescent labeling of each protein species across the proteome. Here, we developed a method to evaluate the labeling homogeneity across the proteome based on a single molecule fluorescence imaging assay. In our measurement using a HeLa cell sample, the proportion of proteins labeled with at least one dye, which we termed &#8216;labeling occupancy&#8217; (LO), was determined to range from 50% to 90%, supporting the high potential of the application of single molecule imaging to sensitive and precise proteome analysis.

Here, we present a protocol to assess the labeling homogeneity for each protein species in a complex protein sample at the single molecule level.

Cuantificación de todo el proteoma de etiquetado homogeneidad en el nivel de molécula única

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

Análisis de tiempo de permanencia de una sola molécula de la escisión de ADN mediada por endonucleasas de restricción

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

Realización de mediciones FRET precisas y precisas de una sola molécula utilizando el smfBox de código abierto

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

La extracción de moléculas de glucógeno hepático para la determinación de la estructura del glucógeno

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

Visualización de la dinámica conformacional de los receptores de membrana utilizando FRET de molécula única

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

Recubrimiento de rejillas CryoEM con grafeno monocapa para mejorar la preparación de muestras criogénicas

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Autor destacado: Mejora de la preparación de muestras de CryoEM mediante monocapa de grafeno en rejillas de microscopía 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

Aplicación de grafeno monocapa a rejillas de criomicroscopía electrónica para la determinación de estructuras de alta resolución

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...