カルシウムイオン反応性DNA結合は,亜鉛指融合タンパク質に結合する
Akira Onoda1, Nozomi Arai, Naoto Shimazu
1Department of Chemistry, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo, 162-8601, Japan.
Journal of the American Chemical Society
|November 25, 2005
まとめ
研究者は,DNA結合制御のために,カルシウム反応性亜鉛指タンパク質を設計した. これらの新しい融合タンパク質は,カルシウムイオン濃度によって調節される調節可能なDNA結合親和性を示し,高度な人工転写因子への道を開く.
科学分野:
- バイオケミストリー バイオケミストリー
- 分子生物学は分子生物学である.
- プロテイン工学は,タンパク質の
背景:
- 亜鉛指タンパク質は,DNA認識と遺伝子調節に不可欠です.
- 制御可能なDNA結合タンパク質の開発は,合成生物学と遺伝子治療にとって不可欠です.
- カルシウムイオンは,タンパク質の機能を調節する生物学的スイッチとして使用することができます.
研究 の 目的:
- カルシウム反応性DNA結合能力を持つ新しい亜鉛指融合タンパク質を設計する.
- これらの融合タンパク質の構造的整合性と折り畳みを調査する.
- DNA結合親和性のCa依存変調を評価する.
主な方法:
- 構造分析のための紫外線光譜検査と円形二重化 (CD) 実験.
- 詳細な折り畳み研究のための核磁気共鳴 (NMR) スペクトロスコピーと構造計算.
- 異なったCa2+濃度下でのDNA結合親和性を定量化するために,電離運動シフトアッセイ (EMSA) を用いる.
主要な成果:
- 融合タンパク質F2-Tnは,統合されたCa-結合ドメインを持つネイティブの亜鉛指の折りたたみを示した.
- 2本の指のトロポニン融合タンパク質 (F1F2-Tn) は,Ca-応答性DNA結合を示した.
- F1F2-Tnの解離定数 (Kd) は,Ca2+なしでは5.8 nM,Ca2+等価100個で13 nMであった.
結論:
- Ca-結合ドメインを組み込んだ人工的に設計された融合亜鉛指タンパク質は,Ca-反応性DNA結合を示す.
- この研究は,カルシウムスイッチによる亜鉛指ベースの人工転写因子を構築するための基礎を提供します.
- エンジニアリングされたタンパク質は,精密で外部から制御される遺伝子調節のための新しいメカニズムを提供します.
関連する概念動画
02:16Transcription Factors
Tissue-specific transcription factors contribute to diverse cellular functions in mammals. For example, the gene for beta globin, a major component of hemoglobin, is present in all cells of the body. However, it is only expressed in red blood cells because the transcription factors that can bind to the promoter sequences of the beta globin gene are only expressed in these cells. Tissue-specific transcription factors also ensure that mutations in these factors may impair only the function of...
01:49Conserved Binding Sites
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 analyses the...
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 analyses the...
02:13Cooperative Binding of Transcription Regulators
Transcriptional regulators bind to specific cis-regulatory sequences in the DNA to regulate gene transcription. These cis-regulatory sequences are very short, usually less than ten nucleotide pairs in length. The short length means that there is a high probability of the exact same sequence randomly occurring throughout the genome. Since regulators can also bind to groups of similar sequences, this further increases the chances of random binding. Transcriptional regulators form dimers that...
02:42Eukaryotic Transcription Activators
Transcription activators are proteins that promote the transcription of genes from DNA to RNA. In most cases, these proteins contain two separate domains ‒ a domain that binds to DNA and a domain for activating transcription; however, in some cases, a single domain is responsible for both binding and activation of transcription, as seen in the glucocorticoid receptor and MyoD.
The binding domains are capable of recognizing and interacting with regulatory sequences on the DNA. These domains are...
The binding domains are capable of recognizing and interacting with regulatory sequences on the DNA. These domains are...
01:27Feedback Regulation of Calcium Concentration
Calcium is an essential signaling molecule required for various cellular functions. Calcium pumps and ion channels on cell and organellar membranes, such as those on the endoplasmic reticulum (ER), regulate calcium concentrations inside the cell. They remain closed, keeping the cytosolic calcium levels low at a resting state.
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
Various transmembrane receptors, such as G protein-coupled receptors (GPCRs), elicit a response to extracellular signals by increasing cytosolic calcium. Activated GPCRs...
01:16Calmodulin-dependent Signaling
Calmodulin (CaM) is a calcium-binding protein in eukaryotes that controls various calcium-regulated cellular processes. It has four calcium-binding sites that bind calcium to form the calcium-calmodulin ( Ca2+-CaM) complex. GPCR stimulation increases the calcium levels in the cells that bind to CaM and induces a conformational change.
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...
The Ca2+-CaM complex does not have enzymatic activity by itself. Instead, the complex binds downstream target proteins, including membrane proteins or enzymes,...