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

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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).