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

Na célula, as proteínas colidem aleatoriamente com outras moléculas, tais como outras proteínas, ácidos nucleicos, e pequenas moléculas ligantes. Se a ligação for não-específica, poucas interações não-covalentes entre as moléculas resultam em uma breve associação. Se um ligante específico ligar à proteína, forma interações não-covalentes extensivas ao longo de superfícies complementares.Tais complexos são estáveis permanecendo ligados por um longo tempo antes de dissociar. A força de uma ligação vinculante é relatada em termos de sua constante de equilíbrio, Kb, também chamada de constante de ligação ou associação. Kb pode ser calculada a partir da relação da concentração do complexo proteína-ligante sobre as concentrações de proteína não ligada e ligante encontrado no equilíbrio.Dada a sua relação com a mudança de energia livre devido à ligação, um grande Kb significa uma grande diminuição no delta G indicando forte afinidade entre proteína e ligante. Dois processos concorrentes são importantes para ligação de proteína-ligante. A associação de uma proteína e um ligante para formar um complexo e a dissociação do complexo nos reagentes.A constante de associação K em é uma medida do número de eventos de ligação por segundo entre uma proteína e seu ligante. Pode ser usada para calcular a taxa de ligante ligação à proteína em uma determinada concentração. inversamente, K off é uma medida do número de eventos de dissociação.Pode ser usada para calcular a rapidez com que o complexo se separa quando a taxa de associação é igual à taxa de dissociação e o equilíbrio é alcançado, onde as concentrações de produtos e reagentes permanecem constantes. Assim, no equilíbrio, K vezes o produto das concentrações de equilíbrio da proteína e do ligante, iguala K off vezes a concentração de equilíbrio a concentração do complexo de proteína de ligante Rearranjando esta expressão mostra também que a relação de K sobre a K off iguala Kb no equilíbrio.

<ol>
	<li>Pollard, Thomas D. "Um guia para ensaios de ligação simples e informativos." Biologia molecular da célula 21, nº 23 (2010): 4061-4067.</li>
	<li>Hulme, Edward C. e Mike A. Trevethick. "Ensaios de ligação de ligantes em equilíbrio: validação e interpretação." Jornal britânico de farmacologia 161, nº 6 (2010): 1219-1237.</li>
	<li>Medina-Franco, José L., Oscar Méndez-Lucio e Karina Martinez-Mayorga. "A interação entre modelagem molecular e quimioinformática para caracterizar paisagens de interações proteína-ligante e proteína-proteína para descoberta de medicamentos." Em Avanços em química de proteínas e biologia estrutural, vol. 96, pp. 1-37. Imprensa Acadêmica, 2014.</li>
</ol>

a constante de ligação de equilíbrio e a força de ligação

The Equilibrium Binding Constant and Binding Strength

A constante de ligação de equilíbrio (Kb) quantifica a força de uma interação proteína-ligante. Kb pode ser calculada da seguinte forma quando a reação está em equilíbrio: <img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center;"> onde P e L são a proteína e o ligante não ligados, respectivamente, e PL é o complexo proteína-ligante. Como a quantidade de ligante ligado também está relacionada à taxa de ligação do ligante, os experimentos também podem determinar Kb examinando as taxas de associação proteína-ligante (kon) e dissociação (koff) usando a seguinte proporção: <img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center;"> Assim, duas categorias de ensaios de ligação são usadas para determinar a constante de ligação de equilíbrio – aqueles que medem as concentrações de equilíbrio e aqueles que medem a cinética de uma reação. Nesse caso, a reação deve estar em equilíbrio no momento da medição. O método de determinação das concentrações de equilíbrio depende da sensibilidade desejada e da facilidade de detecção do sinal. Por essa razão, os ensaios espectroscópicos são os mais amplamente utilizados. Nesses experimentos, a reação produz uma alteração na absorbância de um reagente ou produto em determinado comprimento de onda, detectável por um espectrofotômetro UV-Vis. Alternativamente, o reagente ou produto pode ser marcado com uma sonda fluorescente ou pode conter um fluoróforo intrínseco. Então, o progresso da reação pode ser medido a partir da mudança na fluorescência. Esses ensaios são realizados variando as concentrações de um reagente enquanto o resto do experimento é mantido constante. Os resultados podem então ser representados graficamente e analisados com vários métodos de ajuste de curvas. As interações entre proteínas e ligantes também são estudadas usando uma variedade de técnicas bioquímicas e espectroscópicas. A análise estrutural, utilizando cristalografia de raios X e espectroscopia de RMN, auxilia na previsão de interações proteína-ligante por meio de simulações moleculares. Abordagens teóricas e computacionais, como estudos de acoplamento proteína-ligante, são amplamente utilizadas para caracterizar a posição e as interações de ligantes de moléculas pequenas, incluindo candidatos a medicamentos. O projeto de medicamentos auxiliado por computador é uma alternativa rápida e de baixo custo para acelerar o ritmo dos testes convencionais de medicamentos por tentativa e erro.

A Constante de Ligação de Equilíbrio e a Força de Ligação

A constante de ligação de equilíbrio (Kb) quantifica a força de uma interação proteína-ligante. Kb pode ser calculado da seguinte forma quando a reação está em ...

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

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JoVE Core

Cell Biology

Biologia Celular

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Energy and Catalysis

Energia e Catálise

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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 - Biologia Celular ilustra processos celulares complexos em vídeos de animação fáceis de entender e, ao mesmo tempo, abrangentes. Esta coleção foca nos componentes e funções essenciais das células, incluindo organelas e membranas, transporte celular e energia celular. É complementado por vídeos de Cientistas em ação que demonstram a aplicação da biologia celular em experimentos de pesquisa do mundo real.

How Equilibrium Binding Constant Affects Binding

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