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来检测和表征多肽和蛋白质的罕见(激发)状态，这些多肽和蛋白质可能参与生理和病理生理聚合/解聚过程。作为具有高度生理意义的例子，选择了真核细胞骨架肌动蛋白和微管蛋白这两种在细胞中形成微丝和微管的主要聚合物。作为病理生理聚合/聚集过程的例子，将继续研究与阿尔茨海默病和2型糖尿病的发生有关的两种多肽Abeta(β淀粉样蛋白)和IAPP(胰岛淀粉样蛋白形成多肽)。使用高压核磁共振波谱将从结构和热力学上表征这些蛋白不同构象之间的平衡，并与聚合-解聚过程相关。在肌动蛋白和微管蛋白细丝中，受调控的快速聚合和解聚对于细胞的正常功能至关重要。它将通过高压核磁共振谱结合更复杂的核磁共振方法进行表征，如CEST和弛豫色散谱。当疾病发展时，Abeta和IAPP形成淀粉样沉积。15N和13C富集肽的使用将极大地增加信息量。高压核磁共振波谱将被用来描绘自由能格局，并应提供信息，如果罕见的构象状态参与病理聚合。它们的存在可能为内源性变构抑制剂的开发开辟新的途径。通过压力跳跃光谱分析，将更详细地研究解聚反应的动力学。该联盟的合作伙伴提供的高压荧光和红外光谱等其他生物物理方法将补充这些蛋白质的特征。由于可承受高达300兆帕压力的陶瓷电池现已上市，我们将能够在以前无法获得的压力范围内获得核磁共振数据。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，还将继续对压力激活的小分子催化多肽和折叠体中的构象平衡进行高压核磁共振研究，以合成能够模拟自然产生的多肽的不同功能状态的多肽。为了给改进分子动力学计算和用量子化学方法研究压力效应提供实验依据，我们将阐明TMAO等小分子的压力响应。在这里，还将详细研究宇宙亲和性化合物可能的作用模式，特别是它们可能与模型多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.