Structural and Functional Analysis of Silica Forming Organic Matrices

硅藻生物二氧化硅是一种有机-无机杂化材料，其有机成分被认为在生物矿物形态发生中起作用。有机成分可分为两类。首先是可溶性组分，即完全脱矿后可溶于水溶液的分子；其次是不溶性有机基质，脱矿后仍不溶。硅藻藻含有三种不同类型的不溶性有机基质：几丁质网状结构、有机微环和有机微板。在之前的资助期间，我们研究了(i)有机微环和微板的生化组成（与Shevchenko小组合作，SP-3）， （ii）不溶性有机微环的结构和组装机制（与Schlierf小组合作，SP-5），以及（iii）使用可溶性成分和不溶性有机基质重建二氧化硅形态发生。这导致了环状蛋白样和新型有机基质蛋白的鉴定，并揭示了低ph诱导的混合环状蛋白簇的组装及其二氧化硅沉淀活性。值得注意的是，我们的实验还证明了使用来自隐环藻的可溶性成分和不溶性有机基质在体外重建硅藻样分层多孔二氧化硅图案的可行性。在新的资助期内，我们将重点研究导致体外分层多孔二氧化硅模式形成的神秘自组装过程，并旨在阐明其潜在的分子机制。这将通过蛋白质分离和表征（与舍甫琴科小组合作，SP-3），超分辨率荧光显微镜（与Schlierf小组合作，SP-5）以及通过分子遗传学产生荧光蛋白标记的有机成分来实现。这些体外分析将揭示与硅藻生物二氧化硅形态发生相关的生物分子和生物分子相互作用。

Diatom biosilica is an organic-inorganic hybrid material, and the organic components are believed to play a role in biomineral morphogenesis. The organic components can be divided into two groups. Firstly, the soluble components, which are molecules that are soluble in aqueous solution after complete demineralization, and secondly, the insoluble organic matrices, which remain insoluble after demineralization. The diatom Thalassiosira pseudonana contains three different types of insoluble organic matrices: chitin-based meshworks, organic microrings, and organic microplates. During the previous funding period we have investigated (i) the biochemical composition of organic microrings and microplates (collaboration with the Shevchenko group, SP-3), (ii) the structure and assembly mechanism of the insoluble organic microrings (collaboration with the Schlierf group, SP-5), and (iii) the reconstitution of silica morphogenesis using soluble components and insoluble organic matrices. This lead to the identification of cingulin-like as well as novel organic matrix proteins, and revealed the low pH-induced assembly of mixed cingulin clusters and their silica precipitation activities. Remarkably, our experiments also demonstrated the feasibility to reconstitute diatom-like hierarchically porous silica patterns in vitro using soluble components and insoluble organic matrices from Cyclotella cryptica.In the new funding period, we will focus on studying the enigmatic self-assembly process that leads to formation of the hierarchically porous silica patterns in vitro, and aim to elucidate the underlying molecular mechanism. This will be approached using protein fractionation and characterization (collaboration with the Shevchenko group, SP-3), super-resolution fluorescence microscopy (collaboration with the Schlierf group, SP-5), and the generation of fluorescent protein-tagged organic components through molecular genetics. These in vitro analyses will reveal biomolecules and biomolecular interactions that are relevant for biosilica morphogenesis in diatoms.