枯草杆菌芽孢衣蛋白自组装的研究

The spore coat of Bacillus subtilis endospore represents unique example of macromolecular assemblies. It is composed of at least 70 different proteins that via weak bond and weak specific interaction arrange into a complex multilayered structure. This highly ordered structure endows the spore with ability to defend itself against extreme environmental conditions, including heat, drought, UV radiation, toxic chemicals and lysozyme treatment, etc. Within the scope of this project will address the fundamental questions about how the spore coat is assembled. This proposal focus on the spore coat proteins with crucial morphogenetic role in the spore coat formation SpoIVA, SpoVID, and CotE, and on the proteins with intermediate morphogenetic role as SafA, CotH and CotO, and also on some outer spore coat proteins CotV, CotW, CotY, CotZ. The interactions and dynamic processes among spore coat proteins will be studied by Atomic force microscopy (AFM) at the single molecule level. The high-resolution AFM imaging is used to monitoring the assembly pathway and dynamics of spore coat proteins in vitro. Combined with UV-VIS spectrum and circular dichroic spectroscopy, dynamic light scattering and surface plasmon resonance, we want to establish a new model for spore coat self-assembly process. The attention will be also brought to the unique self-assembly properties of individual or combined several coat proteins in vitro. We will try to establish a synthetic spore coat assembly system which are resistant to various hard conditions.

枯草杆菌的芽孢衣是生物分子自组装方面的特例，它是由约70 种蛋白通过弱键和弱特异性相互作用形成规则的层状结构，这些规则排列的蛋白赋予芽孢抵抗外界热、干旱、辐射、有毒化学物质和酶分解等属性。本项目拟以芽孢衣形态发生蛋白如SpoIVA、SpoVID和CotE，还有起着中间体作用的芽孢衣形态发生蛋白如SafA、CotH 和CotO，及其它芽孢衣蛋白如CotV、CotW、CotY 和CotZ 为主要研究对象，用原子力显微镜在单分子水平上研究芽孢衣蛋白间的相互作用及动力学过程，模拟并高分辨监控芽孢衣蛋白的自组装过程，同时结合紫外可见光谱、圆二色谱、动态光散射和表面等离子共振等技术深入系统地探索芽孢衣蛋白自组装过程及调控规律，实现单一或复合芽孢衣蛋白在生物体外的可控自组装，建立和发展新的蛋白自组装结构和体系，构筑对外界的有毒物质或不良环境有抵抗力功能的自组装体系。

枯草芽孢杆菌的芽孢衣是由70多种不同的蛋白构成的复杂的高度有序的层状结构，它赋予芽孢极强的抗逆能力。芽孢衣蛋白的自组装已成为研究生物大分子进行自我组装的模型。本项目用原子力显微镜在单分子水平上研究芽孢衣蛋白间的相互作用及动力学过程，我们运用单分子力谱研究芽孢衣形态发生蛋白SpoVID、CotY、CotE、 SpoIVA， SafA和CotZ之间的特异性相互作用。通过对两两蛋白间相互作用皮牛级力的测量，获得一系列蛋白间相互作用的动力学数据。我们利用AFM成像在固液界面研究了CotE与CotZ的组装行为，并在固液界面上构建了CotE、CotZ以及CotE与CotZ蛋白自组装体系。发现当CotE与CotZ以较高的摩尔比混合时，它们易于形成延伸的网络结构，而以较低的摩尔比混合时，则易于形成平行的条纹结构。芽孢衣蛋白，尤其是形态发生蛋白之间的相互作用是精确定位因子，决定着芽孢衣的组装过程。CotE与CotZ间特定相互作用的研究丰富了我们对于芽孢衣蛋白组装机制的看法，该组装机制控制形态发生蛋白构建基础支架为其他芽孢衣蛋白的组装提供平台，使我们对芽孢衣的组装和性质的认识越来越深入。