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

В клетке, белки беспорядочно сталкиваются с другими молекулами, такими, как другие белки, нуклеиновые кислоты, и небольшие молекулы лиганды. Если связывание неспецифично, малое число нековалентных взаимодействий между молекулами приводит в краткой ассоциации. Если определенный лиганд связывается с белком, он образует обширные нековалентные взаимодействия вдоль дополняющих поверхностей.Такие комплексы стабильны, и долго сохраняются прежде чем отсоединиться. Сила связывающих взаимодействий сообщается в терминах константы равновесия, Кб, также называемой константа связи или ассоциации. KB можно рассчитать исходя из отношения концентрации белково-лигандового комплекса к концентрациям несвязанных белков и лиганд, найденым в состоянии равновесия.Учитывая ее связь с изменением свободной энергии из-за связывания, Высокое значение Kb означает существенное уменьшение дельта G, указывающее на сильное сродство между белком и лигандом. Два конкурирующих процесса важны для связывания типа белок-лиганд. Ассоциация белка и лиганда для сформировния комплекса и диссоциации комплекса в реактанты.Константа связи K_on это мера числа связываний в секунду между белком и его лигандом. Её можно использовать для расчета скорости связывания лиганда с белком при данной концентрации. И наоборот, K_off это мера числа диссоциаций.Его можно использовать для расчета, как быстро комплекс развалится, когда скорость ассоциации равна скорости диссоциации и достигнуто равновесие, при чём концентрации продуктов и реактантов остаются постоянными. Таким образом, в равновесии, K_on умножить на продукт при равновесных концентрациях белка и лиганда, равен K_off умножиьт на равновесную концентрацию белково-лигандового комплекса. Переупорядочивание этого выражения также показывает, что отношение K_ON к K_OFF равно Kb в равновесии.

<ol>
	<li>Поллард, Томас Д. "Руководство по простым и информативным анализам связывания." Молекулярная биология клетки 21, No23 (2010): 4061-4067.</li>
	<li>Хьюм, Эдвард К., и Майк А. Треветик. "Анализы связывания лигандов в равновесии: валидация и интерпретация." Британский журнал фармакологии 161, No6 (2010): 1219-1237.</li>
	<li>Медина-Франко, Хосе Л., Оскар Мендес-Лусио и Карина Мартинес-Майорга. "Взаимодействие между молекулярным моделированием и хемоинформатикой для характеристики ландшафтов белок-лигандных и белок-белковых взаимодействий для открытия лекарств." В книге "Достижения в химии белка и структурной биологии", том 96, стр. 1-37. Академическая пресса, 2014.</li>
</ol>

Равновесная константа связывания и прочность связи

The Equilibrium Binding Constant and Binding Strength

Константа равновесного связывания (KbKb) количественно определяет силу взаимодействия белок-лиганд. Kb можно рассчитать следующим образом, когда реакция находится в равновесии: 
 <img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center;"> 

 где P и L — несвязанный белок и лиганд соответственно, а PL — комплекс белок-лиганд. 

 Поскольку количество связанного лиганда также связано со скоростью связывания лиганда, в экспериментах можно также определить KbKb, исследуя скорости ассоциации белок-лиганд (konkon) и диссоциации (koffkoff), используя следующее соотношение: 

 <img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center;"> 

 Таким образом, для определения равновесной константы связывания используются две категории анализов связывания: те, которые измеряют равновесные концентрации, и те, которые измеряют кинетику реакции. В этом случае реакция должна находиться в равновесии во время измерения. 

 Метод определения равновесных концентраций зависит от желаемой чувствительности и простоты обнаружения сигнала. По этим причинам наиболее широко используются спектроскопические анализы. В этих экспериментах реакция вызывает изменение оптической плотности реагента или продукта при заданной длине волны, которое можно обнаружить с помощью УФ-ВИД-спектрофотометра. Альтернативно, реагент или продукт могут быть помечены флуоресцентным зондом или могут содержать собственный флуорофор. Затем ход реакции можно измерить по изменению флуоресценции. Эти анализы проводятся путем изменения концентрации одного реагента, в то время как остальная часть эксперимента остается постоянной. Затем результаты можно отобразить на графике и проанализировать с помощью различных методов аппроксимации кривой. 

 Взаимодействия между белками и лигандами также изучаются с использованием различных биохимических и спектроскопических методов. Структурный анализ с использованием рентгеновской кристаллографии и ЯМР-спектроскопии помогает прогнозировать взаимодействия белок-лиганд посредством молекулярного моделирования. Теоретические и вычислительные подходы, такие как исследования докинга белков и лигандов, широко используются для характеристики положения и взаимодействия лигандов малых молекул, включая кандидаты в лекарственные препараты. Компьютерный дизайн лекарств — это быстрая и недорогая альтернатива ускорению традиционных испытаний лекарств методом проб и ошибок.

Равновесная константа связывания (Kb) количественно определяет силу белок-лигандного взаимодействия. Kb можно рассчитать следующим образом, когда реакция находится на уровне ...

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

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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 Cell Biology illustrates complex cellular processes in easy-to-understand yet comprehensive animated video lessons. This collection focuses on the essential components and functions of cells, including organelles and membranes, cellular transport, and cell energetics. It is complemented by scientist-in-action videos that demonstrate the application of cell biology in real-world research experiments.

How Equilibrium Binding Constant Affects Binding

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