Exploring thermodynamic and functional properties of a nucleotide binding protein intracellularly using high resolution fluorine NMR spectroscopy

使用高分辨率氟核磁共振波谱探索细胞内核苷酸结合蛋白的热力学和功能特性

• 批准号: 441495846
• 负责人: Professor Dr. Michael Kovermann
• 金额: --
• 依托单位: Arbeitsgruppe Magnetische Resonanzspektroskopie (NMR)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/441495846?language=en
• 关键词: Exploring; thermodynamic; functional; properties; nucleotide

High-resolution NMR spectroscopy comprises a variety of tools to probe structural, dynamic and functional features of proteins. Most studies which have been conducted so far have been performed under dilute conditions („in vitro“) neglecting the natural environment proteins are exposed to. Therefore the present proposal aims to quantify the thermodynamic stability, intrinsic dynamics and ligand binding affinity of a protein under both, cell lysate and intracellular conditions. It is proposed to label the cold shock protein - which inherently possesses a nucleotide binding site - with fluorine and to subsequently study the protein in its native environment. Using this labeling strategy enables to blend out the large signal background arising from proton, nitrogen and carbon resonances efficiently. Two-dimensional heteronuclear NMR experiments using fluorine (representing the protein) and phosphorus (representing the ligand) as well as fluorine and carbon (representing isotopically labeled ligand) are plannend to be applied to determine the affinity between the nucleotide and the protein in cell lysate and in an intracellular context. Thus the proposed work aims in parallel to contribute to the open question if and how the general properties of a protein like structure, dynamics and function differ when comparing dilute with cellular conditions. The determination of the affinity between a protein and a ligand in a cellular context possesses in this context an ideal potential in order to expand the application of multidimensional NMR correlation experiments comprising fluorine resonances significantly.

高分辨率NMR光谱学包括多种工具来探测蛋白质的结构、动力学和功能特征。到目前为止，大多数研究都是在稀释条件下（“体外”）进行的，忽略了蛋白质暴露于的自然环境。因此，本发明的目的是量化蛋白质在细胞裂解物和细胞内条件下的热力学稳定性、内在动力学和配体结合亲和力。有人建议用氟标记冷休克蛋白-它本身具有核苷酸结合位点-并随后在其天然环境中研究该蛋白质。使用这种标记策略能够有效地混合出由质子、氮和碳共振产生的大信号背景。使用氟（代表蛋白质）和磷（代表配体）以及氟和碳（代表同位素标记的配体）的二维异源NMR实验被计划应用于确定细胞裂解物和细胞内环境中核苷酸和蛋白质之间的亲和力。因此，所提出的工作旨在并行地对开放性问题做出贡献，即当比较稀释与细胞条件时，蛋白质的一般性质如结构、动力学和功能是否以及如何不同。在细胞环境中蛋白质和配体之间的亲和力的测定在这种情况下具有理想的潜力，以便显著地扩展包括氟共振的多维NMR相关实验的应用。