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：（一）生物硅中硅/有机界面的结构是什么，在这个界面上存在哪些相互作用？(ii)新型膜蛋白Sin 1的结构是什么？为了回答问题（i），布伦纳小组将在第一个资助期内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和柠檬酸/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）和不溶性基质之间的相互作用）。将采用固态NMR光谱法，必要时采用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（[13 C，15 N，29 Si]富集样品）的光谱研究和体外研究。生物硅的调查将集中在物种Cyclotella cryptica和Thalassiosira dumanana。生物硅来自T.将研究是否存在结构蛋白质。在C. cryptica，不溶性的，含有几丁质的有机基质发现的Kröger组（SP-1）是特别感兴趣的。体外研究将包括使用来自盖耶集团（SP-6）的位点特异性13 C/15 N标记的合成LCPA对LCPA-二氧化硅复合材料进行的扩展研究。将在生理相关条件（pH 5-6，环境温度）下使用29 Si富集的柠檬酸进行硅化。这种复合材料也可以在不溶性有机基质的存在下合成，梭cryptica（有和没有LCPA）。Cuniberti小组（SP-8）进行的计算将提供进一步的见解并帮助解释光谱数据。为了回答问题（ii），Baldus小组将进行研究以确定新型膜蛋白Sin 1的3D结构及其聚集行为。这种蛋白质是由Kröger和Shevchenko小组（SP-1和SP-3）在第一个资助期发现的。固态核磁共振光谱在非常高的领域（目前高达950 MHz的质子共振频率，在不久的将来在1.2 GHz）和-如果必要的话-DNP辅助固态核磁共振光谱（800 MHz）将采用Baldus组，以回答以下问题：可溶性Sin 1聚集体的结构是如何组织的？Sin 1如何与膜相互作用？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).