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

W komórce białka losowo zderzają się z innymi cząsteczkami, takimi jak inne białka, kwasy nukleinowe i ligandy drobnocząsteczkowe.
Jeśli wiązanie jest niespecyficzne, kilka niekowalencyjnych oddziaływań między cząsteczkami powoduje krótkie skojarzenie.
Jeśli określony ligand wiąże się z białkiem, tworzy rozległe oddziaływania niekowalencyjne wzdłuż komplementarnych powierzchni. Takie kompleksy są stabilne, pozostają związane przez długi czas, zanim się rozłączą.
Siła oddziaływania wiązania jest podawana w postaci jego stałej równowagi, Kb, zwanej również stałą wiązania lub asocjacji. 
Kb można obliczyć ze stosunku stężenia kompleksu białko-ligand do stężeń białka niezwiązanego i liganda znajdujących się w stanie równowagi. 
Biorąc pod uwagę jego związek ze zmianą energii swobodnej spowodowaną wiązaniem, duże Kb oznacza duży spadek delta G, co wskazuje na silne powinowactwo między białkiem a ligandem.
Dwa konkurencyjne procesy są ważne dla wiązania białka-liganda: asocjacja białka i liganda w celu utworzenia kompleksu oraz dysocjacja kompleksu na reagenty.
Stała asocjacji, kon, jest miarą liczby zdarzeń wiązania na sekundę między białkiem a jego ligandem; może być użyta do obliczenia szybkości wiązania liganda z białkiem przy danym stężeniu. 
I odwrotnie, koff jest miarą liczby zdarzeń dysocjacji; może być użyta do obliczenia, jak szybko kompleks się rozpada.
Gdy szybkość asocjacji jest równa szybkości dysocjacji, osiągana jest równowaga, w której stężenia netto produktów i reagentów pozostają stałe.
Zatem w stanie równowagi krazy iloczyn stężeń równowagowych białka i liganda jest równy k razy stężenie równowagowe kompleksu białko-ligand.
Przegrupowanie tego wyrażenia pokazuje również, że stosunek kon do koff jest równy Kb w stanie równowagi.

<ol>
	<li>Pollard, Thomas D. "Przewodnik po prostych i pouczających testach wiązania". Biologia molekularna komórki 21, nr 23 (2010): 4061-4067.</li>
	<li>Hulme'a, Edwarda C. i Mike'a A. Trevethicka. "Testy wiązania ligandów w stanie równowagi: walidacja i interpretacja". Brytyjski dziennik farmakologiczny 161, nr 6 (2010): 1219-1237.</li>
	<li>Medina-Franco, José L., Oscar Méndez-Lucio i Karina Martinez-Mayorga. "Wzajemne oddziaływanie między modelowaniem molekularnym a chemoinformatyką w celu scharakteryzowania krajobrazów interakcji białko-ligand i białko-białko w celu odkrywania leków". W: Postępy chemii białek i biologii strukturalnej, tom 96, s. 1-37. Wydawnictwo Naukowe, 2014.</li>
</ol>

stała wiązania równowagi i siła wiązania

The Equilibrium Binding Constant and Binding Strength

Stała wiązania (kb) określa ilościowo siłę oddziaływania białko-ligand. Gdy reakcja jest w równowadze, kb można obliczyć w następujący sposób:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center;">

gdzie P i L oznaczają odpowiednio niezwiązane białko i ligand, a PL oznacza kompleks białko-ligand.

Ponieważ ilość związanego liganda jest również powiązana z szybkością wiązania ligandu, eksperymenty mogą również określić kb poprzez zbadanie szybkości asocjacji białko-ligand (kon) i dysocjacji (koff) przy użyciu następującego stosunku:

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center;">

Zatem do określenia stałej równowagi wiązania stosuje się dwie kategorie testów wiązania – te, które mierzą stężenia równowagowe i te, które mierzą kinetykę reakcji. W takim przypadku reakcja musi być w równowadze w momencie pomiaru.

Metoda wyznaczania stężeń równowagowych zależy od pożądanej czułości i łatwości detekcji sygnału. Z tych powodów najczęściej stosuje się testy spektroskopowe. W tych eksperymentach reakcja powoduje zmianę absorbancji reagenta lub produktu przy danej długości fali, wykrywalną za pomocą spektrofotometru UV-Vis. Alternatywnie, reagent lub produkt można oznaczyć sondą fluorescencyjną lub może zawierać wewnętrzny fluorofor. Następnie postęp reakcji można zmierzyć na podstawie zmiany fluorescencji. Testy te przeprowadza się poprzez zmianę stężeń jednego reagenta, podczas gdy pozostała część eksperymentu pozostaje stała. Wyniki można następnie przedstawić na wykresie i przeanalizować za pomocą różnych metod dopasowywania krzywych.

Interakcje między białkami i ligandami bada się również przy użyciu różnych technik biochemicznych i spektroskopowych. Analiza strukturalna wykorzystująca krystalografię rentgenowską i spektroskopię NMR pomaga w przewidywaniu interakcji białko-ligand za pomocą symulacji molekularnych. Podejścia teoretyczne i obliczeniowe, takie jak badania dokowania białko-ligand, są szeroko stosowane w celu scharakteryzowania pozycji i interakcji ligandów małocząsteczkowych, w tym kandydatów na leki. Wspomagane komputerowo projektowanie leków to szybka i tania alternatywa umożliwiająca przyspieszenie tempa konwencjonalnego testowania leków metodą prób i błędów.

Stała wiązania równowagi i siła wiązania

Stała wiązania równowagi (Kb) określa ilościowo siłę oddziaływania białko-ligand. Kb można obliczyć w następujący sposób, gdy reakcja zachodzi w ...

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

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

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How Equilibrium Binding Constant Affects Binding

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