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

細胞内では、タンパク質は他のタンパク質、核酸、低分子リガンドなどの他の分子とランダムに衝突します。
結合が非特異的である場合、分子間の非共有結合相互作用はほとんどなく、短い会合を生じます。
特定のリガンドがタンパク質に結合すると、相補的な表面に沿って広範な非共有結合性相互作用が形成されます。そのような複合体は安定しており、解離する前に長期間結合したままである。
結合相互作用の強度は、その平衡定数K b(結合定数または会合定数とも呼ばれる)の観点から報告されます。 
Kbは、平衡状態で見出される未結合タンパク質およびリガンドの濃度に対するタンパク質-リガンド複合体の濃度の比から計算できます。 
結合による自由エネルギー変化との関係を考えると、Kbが大きいとデルタGが大幅に減少し、タンパク質とリガンドの親和性が強いことを示しています。
タンパク質-リガンド結合には、タンパク質とリガンドの会合による複合体の形成と、複合体の反応物への解離という2つの競合するプロセスが重要です。
関連定数konは、タンパク質とそのリガンドとの間の1秒あたりの結合イベントの数を示す尺度であり、特定の濃度でのタンパク質へのリガンド結合速度を計算するために使用できる。 
逆に、koff は解離イベントの数を示す尺度であり、複合体がどれだけ早く崩壊するかを計算するために使用できます。
会合速度が解離速度と等しい場合、平衡に達し、生成物と反応物の正味濃度は一定のままです。
したがって、平衡状態では、タンパク質とリガンドの平衡濃度の積のk倍は、タンパク質-リガンド複合体の平衡濃度のk倍に等しくなります。
この式を並べ替えると、kon と koff の比率が平衡状態で Kb に等しいことも示しています。

<ol>
	<li>ポラード、トーマスD.「シンプルで有益な結合アッセイのガイド」 細胞の分子生物学 21、第23号(2010):4061-4067。</li>
	<li>「平衡状態のリガンド結合アッセイ:検証と解釈」 British journal of pharmacology 161, no. 6 (2010): 1219-1237.</li>
	<li>Medina-Franco, José L., Oscar Méndez-Lucio, and Karina Martinez-Mayorga. "The interplay between molecular modeling and chemoinformatics to characterize protein-ligand and protein-protein interactions landscapes for drug discovery." in Advances in protein chemistry and structural biology, vol. 96, pp. 1-37.アカデミックプレス、2014年。</li>
</ol>

平衡結合定数と結合強度

The Equilibrium Binding Constant and Binding Strength

平衡結合定数(K_b)は、タンパク質とリガンドの相互作用の強さを定量化します。反応が平衡にある場合、K_bは次のように計算できます。

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 1.png" style="text-align: center; width: 650px;" />

ここで、PとLはそれぞれ未結合のタンパク質とリガンド、PLはタンパク質-リガンド複合体です。

結合したリガンドの量はリガンドの結合速度にも関連するため、実験では、次の比率を使用してタンパク質とリガンドの会合(k_on)と解離(k_off)の速度を調べることによってK_bを決定することもできます。

<img alt="Eq1" src="/files/ftp_upload/11513/Equation 2.png" style="text-align: center; width: 650px;" />

したがって、平衡結合定数を決定するには、平衡濃度を測定するものと反応速度を測定するものの 2 つのカテゴリーの結合アッセイが使用されます。測定を行う際、反応が平衡状態であることが求められる場合があります。

平衡濃度を決定する方法は、必要な感度とシグナル検出の容易さによって異なります。これらの理由から、分光学的アッセイが最も広く使用されています。これらの実験では、反応により特定の波長で反応物または生成物の吸光度変化が生じ、紫外可視分光光度計で検出できます。あるいは、反応物または生成物は蛍光プローブでタグ付けすることも、固有の蛍光団を含むこともできます。 そして、蛍光の変化から反応の進行状況を測定することができます。これらのアッセイは、実験の残りの部分を一定に保ちながら、1 つの反応物の濃度を変化させることによって実行されます。結果はグラフ化され、さまざまな曲線近似法を使用して分析できます。

タンパク質とリガンド間の相互作用も、さまざまな生化学的および分光学的手法を使用して研究されています。 X 線結晶構造解析と NMR 分光法を使用した構造解析は、分子シミュレーションによるタンパク質とリガンドの相互作用の予測に役立ちます。タンパク質とリガンドのドッキング研究などの理論的および計算的アプローチは、薬剤候補を含む小分子リガンドの位置と相互作用を特徴付けるために広く使用されています。コンピュータ支援による医薬品設計は、従来の試行錯誤による医薬品試験のペースを加速する、迅速かつコスト効率の高い代替手段です。

平衡結合定数(Kb)は、タンパク質-リガンド相互作用の強度を定量化します。Kbは、反応が...

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

Education

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Cell Biology

細胞生物学

Unit 1

ユニット1

Energy and Catalysis

エネルギーと触媒

KEY TERMS AND CONCEPTS

主要用語と概念

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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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 細胞生物学は、複雑な細胞プロセスを、わかりやすく包括的なアニメーション ビデオ レッスンで説明します。このコレクションは、細胞小器官や膜、細胞輸送、細胞のエネルギー学など、細胞の必須コンポーネントと機能に焦点を当てています。これは、実際の研究実験における細胞生物学の応用を実証する科学者による実際のビデオによって補足されています。

How Equilibrium Binding Constant Affects Binding

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