Role of α-β transition in the folding of proteins

α-β转变在蛋白质折叠中的作用

• 批准号: 11694208
• 负责人: GOTO Yuji
• 金额: $ 3.14万
• 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: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694208/
• 关键词: Protein; Protein Folding; Heteronuclear NMR; β-Lactoglobulin; β2-Microglobulin; Fluorescence; Single Molecule Analysis; Amyloid Fibril; 変性

The α-helix to β-sheet(α→β)transition of proteins is a key issue to understand the folding and biological function of a number of proteins. Folding of β-lactoglobulin is a useful model for clarifying the mechanism of the α→β transition because the kinetic intermediate contains non-native α-helical structure. With recombinant bovine β-lactoglobulin A uniformly labeled with ^<15>N and heteronuclear NMR spectroscopy, we analyzed its folding kinetics and dynamics.1. The H/D exchange experiments have been performed on the native state, demonstrating the presence of a stable hydrophobic core.2. To define the structural and dynamic properties of an early folding intermediate in β-lactoglobulin, the kinetics of folding was measured, using ultra-rapid mixing techniques in conjunction with hydrogen exchange labeling probed by heteronuclear NMR.We demonstrated that, in the early kinetic intermediate, the non-native α-helix is formed at the N-terminal region of the molecule.3. We studied the conformation and stability of the various forms of β-lactoglobulin by heteronuclar NMR.We showed that the modification of the buried thiol group of β2-microglobulin destabilizes the entire parts of the molecule.4. To understand the mechanism of amyloid formation, which is proposed to include the α→β transition, 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 made of β2-microglobulin and its extension process.

蛋白质的α-螺旋→ β-折叠（α→β）转变是理解许多蛋白质折叠和生物学功能的关键问题。β-乳球蛋白的折叠是阐明α→β转变机制的有用模型，因为动力学中间体含有非天然α-螺旋结构。利用~ 1H ~+ N均匀标记的重组牛β-乳球蛋白A<15>和异位核磁共振谱，分析了其折叠动力学和动力学.在天然状态下进行了H/D交换实验，证明了稳定疏水核的存在.为了确定β-乳球蛋白早期折叠中间体的结构和动力学性质，采用超快速混合技术和氢交换标记技术，结合异源NMR，研究了β-乳球蛋白早期折叠的动力学过程，证明了在早期动力学中间体中，非天然α-螺旋在分子的N-末端区域形成.通过异核核磁共振研究了β-乳球蛋白的构象和稳定性，发现β2-微球蛋白的巯基修饰使整个分子不稳定.为了理解淀粉样蛋白形成的机制，其中包括α→β转变，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纤维。用荧光显微镜观察由β2-微球蛋白构成的淀粉样纤维及其延伸过程。

项目成果

Sakai, Kazuko: "Conformation and stability of thiol-modified bovine β-lactoglobulin."Protein Science. 9(10). 1719-1729 (2000)

Sakai，Kazuko：“硫醇修饰的牛 β-乳球蛋白的构象和稳定性。”蛋白质科学 9(10) 1719-1729 (2000)。

Hong, D.: "Clustering of fluorine-substituted alcohols as a factor responsible for their marked effects on proteins and peptides."J.Am.Chem.Soc.. 121(37). 8427-8433 (1999)

Hong, D.：“氟取代醇的聚集是其对蛋白质和肽产生显着影响的一个因素。”J.Am.Chem.Soc.. 121(37)。

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)。

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)。

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

Kuwata，K.：“β-乳球蛋白早期折叠事件的结构和动力学特征”Nature Struct。 8. 151-155 (2001)