State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
J Struct Biol. 2013 Feb;181(2):128-35. doi: 10.1016/j.jsb.2012.11.002. Epub 2012 Nov 22.
The capability of Bacillus subtilis spores to withstand extreme environmental conditions is thought to be conferred especially by their outermost proteinaceous protective layer, called the spore coat. Of the over 70 proteins that form the spore coat, only a small subset of them affect its morphogenesis, they are referred to as morphogenetic proteins. In this study we investigated the interaction between two spore coat morphogenetic proteins SpoVID and CotE. SpoVID is involved in the process of spore surface encirclement by individual coat proteins, these include CotE, which controls the assembly of the outer coat layer. Both proteins were proposed to be recruited to a common protein scaffold, but their direct association has not been previously shown. Here we studied the interactions between CotE and SpoVID in vitro for the first time by using molecule recognition force spectroscopy, which allows the detection of piconewton forces between conjugated biological pairs and also facilitates the investigation of dynamic processes. The most probable CotE-CotE unbinding force was 49.4±0.1pN at a loading rate of 3.16×10³ pN/s while that of SpoVID-CotE was 26.5±0.6pN at a loading rate of 7.8×10² pN/s. We further analyzed the interactions with the bacterial two hybrid system and pull-down experiments, which also indicate that SpoVID interacts directly with CotE. In combination with the previously identified direct contacts among SpoIVA, SpoVID and SafA, our data imply that the physical association of key morphogenetic proteins forms a basic skeleton where other coat proteins could be attached.
枯草芽孢杆菌孢子能够耐受极端环境条件的能力被认为主要归因于其最外层的蛋白质保护性外层，称为孢子壳。在形成孢子壳的超过 70 种蛋白质中，只有一小部分影响其形态发生，它们被称为形态发生蛋白。在这项研究中，我们研究了两种孢子壳形态发生蛋白 SpoVID 和 CotE 之间的相互作用。SpoVID 参与了单个外壳蛋白对孢子表面的包围过程，其中包括 CotE，它控制着外壳层的组装。这两种蛋白质都被认为被招募到一个共同的蛋白质支架上，但它们之间的直接关联尚未被证明。在这里，我们首次通过使用分子识别力谱技术在体外研究了 CotE 和 SpoVID 之间的相互作用，该技术允许检测共轭生物对之间的皮牛顿力，并且还便于研究动态过程。在加载速率为 3.16×10³ pN/s 时，最可能的 CotE-CotE 解联合力为 49.4±0.1pN，而在加载速率为 7.8×10² pN/s 时，SpoVID-CotE 的解联合力为 26.5±0.6pN。我们进一步分析了细菌双杂交系统和下拉实验的相互作用，这也表明 SpoVID 与 CotE 直接相互作用。结合先前鉴定的 SpoIVA、SpoVID 和 SafA 之间的直接接触，我们的数据表明，关键形态发生蛋白的物理缔合形成了一个基本骨架，其他外壳蛋白可以附着在该骨架上。
