Characterization of different conformational states of proteins by highpressure NMR spectroscopy

通过高压核磁共振波谱表征蛋白质的不同构象状态

• 批准号: 243247384
• 负责人: Professor Dr. Hans Robert Kalbitzer
• 金额: --
• 依托单位: Institut für Biophysik und physikalische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/243247384?language=en
• 关键词: Characterization; different; conformational; states; proteins

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.

该提案的主要目标是通过高压NMR光谱检测和表征多肽和蛋白质的稀有（激发）状态，这些状态可能涉及生理和病理生理聚合/解聚过程。作为高生理重要性的实例，选择了真核细胞骨架肌动蛋白和微管蛋白的两种主要聚合物，其形成细胞中的微丝和微管。作为病理生理聚合/聚集过程的实例，将继续研究参与阿尔茨海默病和II型糖尿病发展的两种多肽Abeta（β淀粉样蛋白）和IAPP（胰岛淀粉样蛋白形成多肽）。使用高压NMR光谱学，这些蛋白质的不同构象状态之间的平衡将在结构上和化学上表征，并与聚合-解聚过程相关。在肌动蛋白和微管蛋白丝中，受调节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NMR光谱结合更多涉及的NMR方法，如CEST和弛豫色散光谱来表征。当疾病发展时，Abeta和IAPP形成淀粉样蛋白沉积。使用富含15 N和13 C的肽将显著增加信息含量。高压NMR光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一条新的途径。通过压力跃变光谱法，将更详细地研究解聚的动力学。蛋白质的表征将由其他生物物理方法补充，例如由联合体的合作伙伴提供的高压荧光和红外光谱。由于能够承受高达300 MPa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NMR数据。这将大大增加所获得的NMR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NMR研究的构象平衡发生在小的压力激活的催化肽和折叠体将继续，合成多肽，可以模仿不同的功能状态的天然多肽。为了给改进分子动力学计算和压力效应的量子化学方法提供实验基础，将阐明TMAO等小分子的压力响应。在这里，还将详细研究向宇宙性化合物的可能作用模式，特别是它们与模型肽和蛋白质的假定直接相互作用。