保存されたヒスティジン残留は,強化された非典型的に分割されたインテインにおけるエクステイン依存を誘発する
Giridhar Sekar1, Adam J Stevens2, Anahita Z Mostafavi2
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
Journal of the American Chemical Society
|October 4, 2022
まとめ
研究者たちは,隣接するエクステン配列によって引き起こされる制限を克服するために,新しいタンパク質トランスプレイスング (PTS) システム,Cat*を設計しました. この強化されたシステムは,挑戦的なN-エクステン残留物との改善された活動を示し,PTSのアプリケーションを拡張します.
科学分野:
- バイオテクノロジー
- 化学生物学
- 分子生物学
背景:
- スプリット・インテイン媒介型タンパク質トランスプレイスング (PTS) は,バイオテクノロジーにおけるタンパク質結合に不可欠である.
- エクステン配列の隣接するインテインは,スプライシングの効率を阻害し,PTSアプリケーションを制限する.
- "Cat"のような非典型的な分割インテインは,N-エクステイン配列に対して高い感受性を示します.
研究 の 目的:
- 不良なN-エクステイン残留に対する耐性を高める改良された分割インテインシステムを設計する.
- 非典型の分裂インテインにおけるエクステイン依存のメカニズム的基礎を調査する.
- タンパク質工学におけるスプリットインテイン技術の有用性を拡大する.
主な方法:
- 誤差の多いPCRと 細胞ベースの選択を使用して 強化されたインテインを生成する.
- タンパク質の動態を研究するための溶液核磁気共振 (NMR) スペクトロスコーピー.
- 分子ダイナミクス (MD) のシミュレーションで,残留物の相互作用と形状の変化を分析する.
主要な成果:
- 開発された"Cat*"は,問題のあるN-エクステン配列で,PTS活性が著しく強化された人工分離インテインです.
- エクステン依存を媒介するBブロックヒスティジン残基 (His78) の重要な役割を特定した.
- Cat*は,以前に観察されたエクステン関連のスプライシングの制限を克服することが示された.
結論:
- エンジニアリングされたCat*インテインは,非典型的に分割されたインテインのタンパク質結合の適用性を拡大する.
- His78のような重要な残基のダイナミクスを理解することで,エクステインとインテインの相互作用の洞察が得られます.
- この研究は,バイオテクノロジーのための堅固なタンパク質工学のツールの開発を進めています.
関連する概念動画
Conserved Binding Sites
4.3K
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...
4.3K
Tagging and Fusion Proteins
6.8K
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
6.8K
Exon Recombination
3.7K
The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes.
Exon shuffling follows “splice frame rules.” Each exon...
Exon shuffling follows “splice frame rules.” Each exon...
3.7K
Intrinsically Disordered Proteins
18.0K
Intrinsically disordered proteins are a group of proteins that do not fold into specific three-dimensional structures. Their structural flexibility allows them to complement ordered proteins to perform functions that are inaccessible to rigid structures. They are more common in eukaryotes than prokaryotes and may either be exclusively intrinsically disordered or hybrid proteins, consisting of a mix of ordered and disordered regions. The absence of a rigid structure in these proteins can be...
18.0K
Conservation of Protein Domains Over Different Proteins
11.1K
Protein domains are small structurally independent units that are part of a single amino acid chain. Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...
11.1K
Energy to Drive Translocation
2.1K
Mitochondrial protein import is powered by two distinct energy sources: ATP hydrolysis and electrochemical potential across the inner membrane. Newly synthesized precursors are bound by cytosolic chaperones of the Hsp70 family, which guide them to the import receptors on the mitochondrial surface. Utilizing the energy of ATP hydrolysis, Hsp70 chaperones transfer these precursors to the TOM receptors on the mitochondrial outer membrane.
Generally, polypeptides are unfolded by two distinct...
Generally, polypeptides are unfolded by two distinct...
2.1K


