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特定的细胞器中，即二氧化硅沉积囊泡（SDV）。已经发现许多可溶性（硅亲蛋白、硅酸蛋白、长链多胺）以及不溶性生物有机分子（几丁质、扣带蛋白）与生物二氧化硅相关，生物二氧化硅在SDV中形成，然后被胞吐。然而，由于到目前为止还没有开发出从任何二氧化硅形成生物体中分离SDV的方案，因此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。本项目的目的是了解模拟SDV膜的脂质双层对生物分子诱导的二氧化硅形态发生的影响。在上一个资助期间，我们已经成功地建立了由复杂的脂质混合物组成的支持膜，包括硅藻特异性脂质。有了这些膜在手，我们能够分析扣带蛋白，即扣带蛋白W2和扣带蛋白Y3，和长链多胺（LCPA）的相互作用。虽然cingulins不结合这些膜，LCPA与脂质双层相互作用，导致形成三维膜堆叠。通过使用在盖耶组（SP-6）中合成的不同LCPA的系统研究，我们能够表明诱导膜叠层形成需要最小数目的胺基（N > 5）。在即将到来的资助期内，我们将专注于silaffins和SDV膜相关蛋白silicanin-1（Sin 1），这是本研究单位2038的成员在上一个资助期内新发现的。在本研究的第一部分，我们计划在膜中重组表达、分离和重组海链藻的全长Sin 1。我们将调查的组织和自聚集特性的膜限制的Sin 1作为pH值的函数，利用我们已经开发的膜系统，在上一个资助期。此外，在LCPA存在和不存在的情况下，将检查膜限制Sin 1的二氧化硅沉淀特性。为了获得关于Sin 1蛋白的种特异性的信息，我们计划比较来自T.与梭状棘突藻相似。在本提案的第二部分中，我们将分析由盖耶集团（SP-6）提供的具有不同电荷模式的合成硅亲和素1衍生物的膜结合能力和二氧化硅沉淀特性。将结果与用天然硅蛋白1A 1获得的结果进行比较。

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.