Name: how equilibrium binding constant affects binding
Uploaded: 2023-04-30T18:28:41.000Z
Duration: 2 min 18 s
Channel: JoVE

En la célula, las proteínas chocan aleatoriamente con otras moléculas, como otras proteínas, ácidos nucleicos y ligandos de moléculas pequeñas.
Si la unión es inespecífica, pocas interacciones no covalentes entre las moléculas dan lugar a una breve asociación.
Si un ligando específico se une a la proteína, forma extensas interacciones no covalentes a lo largo de superficies complementarias. Tales complejos son estables, permaneciendo unidos durante mucho tiempo, antes de disociarse.
La fuerza de una interacción de enlace se informa en términos de su constante de equilibrio, Kb, también llamada constante de enlace o asociación. 
Kb se puede calcular a partir de la relación entre la concentración del complejo proteína-ligando y las concentraciones de proteína no unida y ligando que se encuentran en equilibrio. 
Dada su relación con el cambio de energía libre debido a la unión, un Kb grande significa una gran disminución en delta G, lo que indica una fuerte afinidad entre la proteína y el ligando.
Dos procesos que compiten entre sí son importantes para la unión proteína-ligando: la asociación de una proteína y un ligando para formar un complejo y la disociación del complejo en los reactivos.
La constante de asociación, kon, es una medida del número de eventos de unión por segundo entre una proteína y su ligando; se puede utilizar para calcular la tasa de unión del ligando a la proteína a una concentración dada. 
Por el contrario, koff es una medida del número de eventos de disociación; se puede usar para calcular qué tan rápido se desintegra el complejo.
Cuando la tasa de asociación es igual a la tasa de disociación, se alcanza un equilibrio, donde las concentraciones netas de productos y reactivos permanecen constantes.
Así, en equilibrio, kpor el producto de las concentraciones de equilibrio de la proteína y el ligando es igual a kpor la concentración de equilibrio del complejo proteína-ligando.
La reorganización de esta expresión también muestra que la relación entre ky koff es igual a Kb en equilibrio.

<ol>
	<li>Pollard, Thomas D. "Una guía para ensayos de unión simples e informativos". Biología molecular de la célula 21, no. 23 (2010): 4061-4067.</li>
	<li>Hulme, Edward C. y Mike A. Trevethick. "Ensayos de unión de ligandos en equilibrio: validación e interpretación". Revista británica de farmacología 161, no. 6 (2010): 1219-1237.</li>
	<li>Medina-Franco, José L., Oscar Méndez-Lucio y Karina Martínez-Mayorga. "La interacción entre el modelado molecular y la quimioinformática para caracterizar los paisajes de interacciones proteína-ligando y proteína-proteína para el descubrimiento de fármacos". En Avances en química de proteínas y biología estructural, vol. 96, pp. 1-37. Editorial Académica, 2014.</li>
</ol>

la constante de unión de equilibrio y la fuerza de unión

The Equilibrium Binding Constant and Binding Strength

La constante de unión de equilibrio (Kb) cuantifica la fuerza de una interacción proteína-ligando. Kb se puede calcular de la siguiente manera cuando la reacción está en equilibrio:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center; max-width: 100%;">

donde P y L son la proteína y el ligando no unidos, respectivamente, y PL es el complejo proteína-ligando.

Como la cantidad de ligando unido también está relacionada con la tasa de unión del ligando, los experimentos también pueden determinar Kb examinando las tasas de asociación proteína-ligando (kon) y disociación (koff) utilizando la siguiente proporción:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center; max-width: 100%;">

Por lo tanto, se utilizan dos categorías de ensayos de unión para determinar la constante de unión en equilibrio: los que miden las concentraciones de equilibrio y los que miden la cinética de una reacción. En este caso, la reacción debe estar en equilibrio en el momento de la medición.

El método para determinar las concentraciones de equilibrio depende de la sensibilidad deseada y la facilidad de detección de la señal. Por estas razones, los ensayos espectroscópicos son los más utilizados. En estos experimentos, la reacción produce un cambio de absorbancia de un reactivo o de un producto a una longitud de onda determinada, detectable mediante un espectrofotómetro UV-Vis. Alternativamente, el reactivo o producto puede marcarse con una sonda fluorescente o puede contener un fluoróforo intrínseco. Entonces, el progreso de la reacción se puede medir a partir del cambio de fluorescencia. Estos ensayos se realizan variando las concentraciones de un reactivo mientras el resto del experimento se mantiene constante. Por tanto, los resultados se pueden representar gráficamente y analizar con varios métodos de ajuste de curvas.

Las interacciones entre proteínas y ligandos también se estudian utilizando una variedad de técnicas bioquímicas y espectroscópicas. El análisis estructural, mediante cristalografía de rayos X y espectroscopia de RMN, ayuda a predecir las interacciones proteína-ligando mediante simulaciones moleculares. Los enfoques teóricos y computacionales, como los estudios de acoplamiento de proteínas y ligandos, se utilizan ampliamente para caracterizar la posición y las interacciones de ligandos de moléculas pequeñas, incluidos los candidatos a fármacos. El diseño de fármacos asistido por computadora es una alternativa rápida y de bajo costo para acelerar el ritmo de las pruebas de fármacos convencionales de prueba y error.

La constante de unión de equilibrio y la fuerza de unión

La constante de unión de equilibrio (Kb) cuantifica la fuerza de una interacción proteína-ligando. Kb se puede calcular de la siguiente manera cuando la reacción es ...

the-equilibrium-binding-constant-and-binding-strength

Education

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Cell Biology

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KEY TERMS AND CONCEPTS

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how equilibrium binding constant affects binding

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions.

This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions.

The page also connects the binding constant to real binding strength. It shows why this value matters when comparing different molecular pairs. For high school students, it provides a clear link between a chemistry concept and molecular behavior.

How Equilibrium Binding Constant Affects Binding

how-equilibrium-binding-constant-affects-binding

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the equilibrium binding constant and binding strength

The equilibrium binding constant describes how strongly two molecules bind at equilibrium. It is a useful way to compare binding strength in molecular interactions. This topic focuses on how binding is measured and what the equilibrium binding constant means. A higher value indicates stronger binding, while a lower value indicates weaker binding. Understanding this constant helps explain how tightly molecules stay together under equilibrium conditions. The page also connects the binding...

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key terms and concepts

Learn how the equilibrium binding constant reflects binding strength and why higher or lower values matter in molecular interactions at equilibrium.

How Equilibrium Binding Constant Affects Binding | JoVE K12

cell biology

JoVE Core Biología Celular ilustra procesos celulares complejos mediante completos vídeos animados fáciles de entender. Esta colección se centra en los componentes esenciales de las células y su funcionamiento, incluyendo los orgánulos y las membranas, el transporte celular y el metabolismo energético celular. Todo este material se complementa con los vídeos de científicos en acción, que muestran la aplicación de la biología celular en experimentos reales de investigación.

How Equilibrium Binding Constant Affects Binding

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