タンパク質標的に緊密に結合するリガンド分子の結合モードを決定するための敏感なNMRアプローチ
Wan-Na Chen1, Christoph Nitsche1,2, Kala Bharath Pilla1
1Australian National University , Research School of Chemistry, Canberra, ACT 2601, Australia.
Journal of the American Chemical Society
|March 15, 2016
まとめ
この研究は,テート-ブチル化学ラベルを用いた新しい核磁気共鳴 (NMR) 方法を導入し,困難な標的に対するタンパク質-リガンド結合モードを決定します. このテクニックは,結晶化が実現できない場合,構造主導の薬剤設計を助けます.
科学分野:
- 生物化学
- 構造生物学
- 薬物の発見
背景:
- タンパク質-リガンド複合体の詳細な知識が必要です.
- タンパク質-リガンド共複合体の結晶化はしばしば困難または不可能である.
- 既存の核磁気共鳴 (NMR) 方法は,緊密に結合するリガンドの研究に限界があります.
研究 の 目的:
- タンパク質を標的とする難易度の高い鉛化合物の結合モードを決定するための敏感なNMRアプローチを開発する.
- 迅速なリガンド交換や安定した同位体ラベリングに依存する現在のNMR技術の限界を克服する.
主な方法:
- 化学的ラベルとして1H NMR信号の狭さと強さを利用した.
- 内部および分子間クロスピークを生成するために,Nuclear Overhauser Effect Spectroscopy (NOESY) を使用した.
- 測定された偽接触は,タンパク質上のランタニドタグからリガンドの位置にシフトする.
- デング熱ウイルスのNS2B-NS3プロテアゼが tert-butylを含むリガンドで複合した方法を実証した.
主要な成果:
- トートブチル群は,異常に強烈な内および分子間NOESYクロスピークを提供しました.
- 偽接触シフトにより,タンパク質上のリガンドの正確な位置づけが可能になりました.
- タンパク質の側鎖共振配分なしで,分子間NOE配分が達成されました.
- デングウイルスのプロテアゼとの高親和性リガンドの結合モードを成功裏に決定した.
結論:
- 提示されたNMRアプローチは,難しいタンパク質のリガンド結合モードの決定に敏感で有効です.
- この方法は結晶化に代わって,既存のNMR技術の限界を克服します.
- 化学ラベルとして tert-ブチル群を使用することで,タンパク質-リガンドシステムに挑戦する構造主導の薬剤設計が容易になります.
関連する概念動画
02:40Ligand Binding Sites
15.7K
Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
15.7K
02:40Ligand Binding Sites
9.1K
9.1K
01:22Protein-Drug Binding: Determination Methods
768
Determining protein-drug binding can be achieved through indirect and direct methods, each providing valuable insights into the interaction between proteins and drugs.
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
Indirect methods involve isolating the bound drug from its free form in biological samples such as blood, serum, or plasma. These techniques aim to measure the percentage of drugs bound to proteins. Equilibrium dialysis is a commonly used method where the free drug concentration at equilibrium is measured by separating the bound...
768
02:18The Equilibrium Binding Constant and Binding Strength
15.6K
The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
15.6K
01:49Conserved Binding Sites
5.3K
Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
5.3K
00:49Ligand Binding and Linkage
5.9K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
5.9K