Silica Nano- to Micro-Patterning at the Membrane Interface in vitro

硅藻中的二氧化硅生物矿化发生在高度指定的细胞器中，即二氧化硅沉积囊泡（sdv）。许多可溶性生物硅（硅油、硅酸、长链多胺）和不溶性生物有机分子（几丁质、环藻肽）都与生物二氧化硅有关，生物二氧化硅在SDV中形成，然后胞外化。然而，由于目前还没有从任何形成二氧化硅的生物体中分离SDV的方案，SDV内发生的过程以及周围脂质膜的组成及其对生物二氧化硅形态发生的影响仍然难以捉摸。本项目旨在了解模拟SDV膜的脂质双分子层对生物分子诱导二氧化硅形态发生的影响。在之前的资助期内，我们已经成功地建立了由复杂的脂质混合物组成的支撑膜，包括硅藻特定的脂质。有了这些膜，我们就能够分析cingulin（即cingulin W2和cingulin Y3）与长链多胺（LCPA）的相互作用。虽然环状蛋白不与这些膜结合，但LCPA与脂质双分子层相互作用，形成三维膜堆。通过对Geyer基团（SP-6）合成的不同LCPA的系统研究，我们能够证明，诱导膜堆栈形成所需的胺基数量最少（N > 5）。在即将到来的资助期内，我们将重点研究silaffins和SDV膜相关蛋白silanin -1 (Sin1)，这是2038研究单元成员在上一个资助期内新发现的。在本课题的第一部分，我们计划重组表达、分离和重组假蓝藻膜中的全长Sin1。我们将利用我们在上一个资助期开发的膜系统，研究膜约束Sin1的组织和自聚集特性作为pH值的函数。此外，我们还将研究在LCPA存在和不存在的情况下，Sin1膜的二氧化硅沉淀性能。为了获得Sin1蛋白的物种特异性信息，我们计划比较假单胞菌（T. pseudonana） Sin1蛋白与fususiformis圆柱体（cylinder drotheca fusformis）的膜特异性。在本论文的第二部分，我们将分析Geyer团队提供的具有不同电荷模式的合成硅粉1衍生物（SP-6）的膜结合能力和二氧化硅沉淀性能。将所得结果与天然矽胶1A1进行比较。

Silica biomineralization in diatoms takes place in highly specified organelles, the silica deposition vesicles (SDVs). A number of soluble (silaffins, silacidins, long chain polyamines) as well as insoluble bioorganic molecules (chitin, cingulins) have been found to be associated with biosilica, which is formed in the SDV and then exocytosed. However, as no protocol has been developed so far to isolate SDVs from any silica-forming organism, the processes occurring within the SDV as well as the composition of the surrounding lipid membrane and its influence on biological silica morphogenesis remain elusive. It is the aim of this project to understand the influence of lipid bilayers mimicking SDV membranes on biomolecule induced silica morphogenesis. In the previous funding period, we have successfully established supported membranes composed of complex lipid mixtures including diatom specific lipids. With these membranes in hand, we were able to analyze the interaction of cingulins, i.e. cingulin W2 and cingulin Y3, and of long-chain polyamines (LCPA). While cingulins do not bind to these membranes, LCPA interact with lipid bilayers resulting in the formation of three-dimensional membrane stacks. By a systematic study using different LCPA synthesized in the Geyer group (SP-6), we were able to show that a minimum number of amine groups (N > 5) is required to induce membrane stack formation. In the upcoming funding period, we will focus on silaffins and the SDV membrane associated protein silicanin-1 (Sin1), newly identified by members of this Research Unit 2038 in the previous funding period. In the first part of this proposal, we plan to recombinantly express, isolate and reconstitute full length Sin1 from Thallasiosira pseudonana in membranes. We will investigate the organization and self-aggregation properties of membrane-confined Sin1 as a function of pH making use of the membrane systems that we have developed in the previous funding period. Moreover, the silica precipitation properties of membrane confined Sin1 in the absence and presence of LCPA will be examined. To obtain information about the species specificity of the Sin1 protein, we plan to compare the membrane specific properties of Sin1 from T. pseudonana with those of Cylindrotheca fusiformis. In the second part of this proposal, we will analyze synthetic silaffin 1 derivatives provided by the Geyer group (SP-6) with different charge patterns with respect to their membrane binding ability and silica precipitation properties. The results will be compared with those obtained with native silaffin 1A1.