Solid-State NMR and DNP Studies of Diatom Biosilica:Organic Matrices and the Silica/Organic Interface

本项目旨在研究两个首要问题：(i)生物二氧化硅中二氧化硅/有机界面的结构是什么？在该界面上存在哪些相互作用？（ii）新型膜蛋白Sin1的结构是什么？为了回答第(1)个问题，Brunner小组将在第一个资助期的发现基础上开展工作。他们将研究有机生物二氧化硅成分与硅酸/二氧化硅之间的相互作用，以及可溶性和不溶性有机生物二氧化硅成分之间的相互作用（例如，长链多胺（LCPA）与不溶性基质之间的相互作用）。固态核磁共振波谱将被采用，如有必要，dnp辅助（与Baldus小组合作）。调查将包括完整的、提取的硅藻生物二氧化硅（[13C,15N，29Si]富集样品）的光谱研究和体外研究。生物二氧化硅的研究将集中在隐环藻和假海藻上。从假单胞菌的生物二氧化硅将研究其结构蛋白的存在。在隐球菌的情况下，Kröger组（SP-1）发现的不溶的，含有几丁质的有机基质是特别感兴趣的。体外研究将包括对LCPA-二氧化硅复合材料的扩展研究，使用来自Geyer组（SP-6）的特定位点13C/ 15n标记的合成LCPA。硅化将在生理相关条件下（pH值5-6，环境温度）使用富含29si的硅酸进行。这种复合材料也可以在不溶性有机基质存在的情况下合成，例如，从隐球菌（含或不含LCPA）合成。由Cuniberti小组（SP-8）进行的计算将提供进一步的见解，并有助于解释光谱数据。为了回答问题（ii）， Baldus团队将进行研究，以确定新型膜蛋白Sin1的3D结构及其聚集行为。这种蛋白质是在第一个资助期由Kröger和舍甫琴科小组（SP-1和SP-3）发现的。高场固态核磁共振波谱（目前高达950 MHz质子共振频率，在不久的将来达到1.2 GHz）和必要时dnp辅助固态核磁共振波谱（800 MHz）将被Baldus团队用于回答以下问题：可溶性Sin1聚集体是如何结构组织的？Sin1是如何与膜相互作用的？同时，将与Kröger小组（SP-3）和Steinem小组（见SP-7项目）密切合作进行研究。

The present project aims to investigate two overarching questions:(i) What is the structure of the silica/organic interface in biosilica and which interactions are present at this interface?(ii) What is the structure of the novel membrane protein Sin1? To answer question (i), the Brunner group will build its work on the discoveries made during the first funding period. They will investigate the interactions between organic biosilica constituents and silicic acid/silica as well as between soluble and insoluble organic biosilica constituents (e.g., the interactions between long-chain polyamines (LCPA) and insoluble matrices). Solid-state NMR spectroscopy will be employed, if necessary DNP-assisted (collaboration with the Baldus group). The investigations will encompass spectroscopic studies of the intact, extracted diatom biosilica ([13C,15N,29Si]-enriched samples) and in vitro studies. The investigation of biosilica will be focused to the species Cyclotella cryptica and Thalassiosira pseudonana. Biosilica from T. pseudonana will be studied with respect to the presence of structured proteins. In the case of C. cryptica, the insoluble, chitin-containing organic matrices found by the Kröger group (SP-1) are of special interest. The in vitro studies will encompass extended investigations of LCPA-silica composites using site-specifically 13C/15N-labeled synthetic LCPA from the Geyer group (SP-6). Silicification will be performed using 29Si-enriched silicic acid under physiologically relevant conditions (pH 5-6, ambient temperature). Such composites will also be synthesized in the presence of insoluble organic matrices, e.g., from C. cryptica (with and without LCPA). Calculations carried out by the Cuniberti group (SP-8) will provide further insight and help interpreting the spectroscopic data.To answer question (ii), the Baldus group will perform studies to determine the 3D structure of the novel membrane protein Sin1 as well as its aggregation behavior. This protein was discovered in the first funding period by the Kröger and Shevchenko groups (SP-1 and SP-3). Solid-state NMR spectroscopy at very high field (currently up to 950 MHz proton resonance frequency, in the near future at 1.2 GHz) and - if necessary - DNP-assisted solid-state NMR spectroscopy (800 MHz) will be employed by the Baldus group in order to answer the following questions: How are soluble Sin1 aggregates structurally organized? How does Sin1 interact with membranes? At the same time, research will be conducted in close collaboration with the Kröger group (SP-3) and the Steinem group (see project SP-7).