Single molecular analysis of protein folding

蛋白质折叠的单分子分析

• 批准号: 10480181
• 负责人: GOTO Yuji
• 金额: $ 8.96万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-10480181/
• 关键词: Protein; Protein Folding; Single Molecule Analysis; β-Lactoglobulin; β2-Microglobulin; Fluorescence; NMR; Amyloid Fibril; 蛋白質

Single molecular analysis is becoming important as a critical approach for characterizing the structure and function of protein molecules. The aim of this project is to improve our understanding of the mechanism of protein folding and stability by using novel approaches of single molecular analysis.1. First, to understand the mechanism of GroEL-assisted protein folding, we studied the interaction of fluorescence-labeled GroEL and substrate proteins at the single molecule level by total internal reflection fluorescence microscopy. We, for the first time, demonstrated the direct interaction between GroEL and substrate proteins, which was dissociated upon addition of ATP.2. To understand the mechanism of amyloid fibril formation, we expressed human β2-microglobulin in the methylotropic yeast, Pichia pastoris. The recombinant β2-microglobulin formed amyloid fibrils as demonstrated by electron microscopy and atomic force microscopy. With fluorescence microscopy, we observed the amyloid fibrils and its extension process.3. We studied the conformation and stability of the various forms of β-lactoglobulin by heteronuclear NMR at the residue level. We showed that the modification of the buried thiol group of β-lactoglobulin destabilizes the entire parts of the molecule.4. An early refolding intermediate of β-lactoglobulin is known to contain non-native α-helical structure. The early stage of β-lactoglobulin refolding was studied using ultra-rapid mixing techniques combined with hydrogen exchange labeling proved by heteronuclear NMR.We demonstrated that, in the kinetic intermediate accumulated at 2 msec of refolding, the non-native α-helix is formed at the N-terminal region of the molecule.

单分子分析作为表征蛋白质分子结构和功能的一种重要方法正变得越来越重要。该项目的目的是通过使用新的单分子分析方法来提高我们对蛋白质折叠机制和稳定性的理解。首先，为了了解GroEL辅助蛋白折叠的机制，我们利用全内反射荧光显微镜在单分子水平上研究了荧光标记的GroEL与底物蛋白的相互作用。我们首次证明了GroEL与底物蛋白之间的直接相互作用，并在加入ATP.2后解离。为了了解淀粉样蛋白纤维形成的机制，我们在嗜甲基酵母毕赤酵母中表达了人β2微球蛋白。电子显微镜和原子力显微镜显示重组β2微球蛋白形成淀粉样蛋白原纤维。用荧光显微镜观察淀粉样蛋白原纤维及其延伸过程。我们用异核磁共振在残馀水平上研究了各种形式的β-乳球蛋白的构象和稳定性。我们发现，对β-乳球蛋白中埋藏的巯基的修饰使分子的整个部分不稳定。已知β-乳球蛋白的早期重折叠中间体含有非天然α-螺旋结构。采用超快速混合技术，结合经核磁共振验证的氢交换标记，研究了β-乳球蛋白重折叠的早期阶段。结果表明，在2 msec重折叠过程中积累的动力学中间体中，非天然α-螺旋在分子的n端区域形成。

项目成果

Kuwata, Kazuo: "Structural and kinetic characterization of early folding events in β-lactoglobulin."Nature Structural Biology. 8(2). 151-155 (2001)

Kuwata, Kazuo：“β-乳球蛋白早期折叠事件的结构和动力学特征。”《自然结构生物学》8(2) 151-155 (2001)。

後藤祐児: "(訳本)タンパク質フォールディングのキネティクス、Bengt Nolting著"シュプリンガーフェアラーク東京. 180 (2000)

Yuji Goto：“（翻译）蛋白质折叠动力学，Bengt Nolting 着”Springer Verlag 东京 180 (2000)。

Kuwata,Kazuo: "Solution structure and dynamics of bovine β-lactoglobulin A"Protein Science. 8(12). 2541-2545 (1999)

Kuwata，Kazuo：“牛 β-乳球蛋白 A 的溶液结构和动力学”蛋白质科学 8(12)。

Kuwata, K.: "α→β Transition of β-lactoglobulin as evidenced by heteronuclear NMR." J.Mol.Biol.283(4). 731-739 (1998)

Kuwata, K.：“异核 NMR 证明了 β-乳球蛋白的 α→β 转变。”J.Mol.Biol.283(4) (1998)。

Hoshino, M.: "High mobility of the phospholipid binding loop of human β2-glycoprotein I domain V revealed by heteronuclear NMR."J.Mol.Biol.. 304(5). 927-940 (2000)

Hoshino, M.：“通过异核 NMR 揭示人 β2-糖蛋白 I 结构域 V 的磷脂结合环的高迁移率。”J.Mol.Biol.. 927-940 (2000)。