Single molecular analysis of protein folding

单分子分析作为表征蛋白质分子结构和功能的重要手段正变得越来越重要。这个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和稳定性机制的理解。首先，为了了解GroEL辅助蛋白质折叠的机制，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与底物蛋白质的相互作用。我们首次证明了GroEL与底物蛋白之间的直接相互作用，这种作用在加入ATP后被解离。为了了解淀粉样蛋白纤维的形成机制，我们在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β-2微球蛋白形成淀粉样纤维。用荧光显微镜观察淀粉样纤维及其延伸过程。我们用核磁共振在残基水平上研究了不同形式的β-乳球蛋白的构象和稳定性。我们发现β-乳球蛋白中埋藏的硫醇基团的修饰破坏了整个分子的稳定性。已知β-乳球蛋白的早期复性中间体含有非天然的α-螺旋结构。用超快速混合技术结合异核核磁共振证明的氢交换标记技术对β-乳球蛋白复性的早期阶段进行了研究。结果表明，在复性时间为2毫秒的动力学中间体中，非天然的α-螺旋形成于分子的N-末端区域。

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.