Characterization of different conformational states of proteins by highpressure NMR spectroscopy

该提案的主要目标是通过高压核磁共振波谱检测和表征可能参与生理和病理生理聚合/解聚合过程的多肽和蛋白质的稀有（激发态）状态。作为高生理重要性的例子，真核细胞骨架的两种主要聚合物肌动蛋白和微管蛋白在细胞中形成微丝和微管。作为病理生理聚合/聚集过程的例子，两种多肽Abeta （β淀粉样蛋白）和IAPP（胰岛淀粉样蛋白形成多肽）的研究将继续进行，这两种多肽与阿尔茨海默病和II型糖尿病的发展有关。利用高压核磁共振波谱法对这些蛋白质不同构象状态之间的平衡进行结构和热力学表征，并与聚合解聚过程有关。在肌动蛋白和微管蛋白丝中，受调节的快速聚合和解聚合对细胞的正常功能至关重要。高压核磁共振波谱将结合更复杂的核磁共振方法，如CEST和弛豫色散波谱进行表征。当疾病发展时，β和IAPP形成淀粉样蛋白沉积。利用富含15N和13C的多肽可以显著提高蛋白质的信息量。高压核磁共振波谱将用于描绘自由能景观，并应提供信息，如果罕见的构象状态涉及病理聚合。它们的存在可能为内在变构抑制剂的开发开辟一条新的途径。通过压力跳变光谱，解聚动力学将得到更详细的研究。蛋白质的表征将由其他生物物理方法补充，如由联盟合作伙伴提供的高压荧光和红外光谱。由于承受压力高达300 MPa的陶瓷电池现在已经商业化，我们将能够在以前无法获得的压力范围内获得NMR数据。这将大大增加所获得的核磁共振数据的信息量，并提高所获得的热力学参数的准确性。此外，高压核磁共振对小压力激活的催化多肽和折叠体中发生的构象平衡的研究将继续进行，合成的多肽可以模拟自然存在的多肽的不同功能状态。为了为分子动力学计算的改进和压力效应的量子化学方法提供实验依据，本文将对TMAO等小分子的压力响应进行阐述。本文还将详细研究宇宙向性化合物可能的作用方式，特别是它们与模型肽和蛋白质的直接相互作用。

The main goal of the proposal is to detect and characterize rare (excited) states of polypeptides and proteins by high-pressure NMR spectroscopy which probably are involved in physiological and pathophysiological polymerization/depolymerisation processes. As examples of high physiological importance, the two main polymers of the eukaryotic cytoskeleton actin and tubulin forming microfilaments and microtubules in the cell were selected. As examples for pathophysiological polymerisation/aggregation processes, the study of the two polypeptides Abeta (beta amyloid) and IAPP (islet amyloid forming polypeptide) will be continued that are involved in the development of Alzheimer disease and diabetes mellitus type II. Using high pressure NMR spectroscopy equilibria between different conformational states of these proteins will be characterized structurally and thermodynamically and related to the polymerizationdepolymerization process. In actin und tubulin filaments, the regulated fast polymerization and depolymerisation is crucial for a proper function of the cell. It will be characterized by high pressure NMR spectroscopy combined with more involved NMR methods such as CEST and relaxation dispersion spectroscopy. Abeta and IAPP form amyloid deposits when the diseases develop. The use of 15N and 13C enriched peptides will increase the information content dramatically. High pressure NMR spectroscopy will be used to delineate the free energy landscape and should give information if rare conformational states are involved in the pathological polymerisation. Their existence may open a new way for the development of intrinsic allosteric inhibitors. By pressure-jump spectroscopy, the kinetics of the depolymerisation will be studied in more detail. The characterization of the proteins will be supplemented by other biophysical methods such as high pressure fluorescence and infrared spectroscopy provided by the partners of the consortium. Since ceramic cells that withstand pressures up to 300 MPa are now commercially available, we will be able to get NMR data in a pressure range not accessible to us before. This will increase dramatically the information content of the obtained NMR data and increase the accuracy of the obtained thermodynamic parameters. In addition, the high-pressure NMR study of the conformational equilibria occurring in small pressure-activated catalytical peptides and foldamers will be continued, synthetic polypeptides that can mimic different functional states of naturally occurring polypeptides. For providing an experimental basis for the improvement of molecular dynamics calculations and quantum chemical approaches to pressure effects, the pressure response of small molecules such as TMAO will be elucidated. Here, also the possible mode of action of cosmotropic compounds will be studied in detail, especially their putative direct interactions with model peptides and proteins.