Investigation of site-specific dynamic nuclear polarization on biomolecules

生物分子位点特异性动态核极化的研究

• 批准号: 232503709
• 负责人: Professor Dr.-Ing. Björn Corzilius
• 金额: --
• 依托单位: Arbeitsgruppe NMR-Festkörperspektroskopie
• 依托单位国家: 德国
• 项目类别: Independent Junior Research Groups
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232503709?language=en
• 关键词: Investigation; site; specific; dynamic; nuclear

During the initial Emmy Noether project it was possible to investigate the relevant mechanisms for dynamic nuclear polarization (DNP) under magic-angle spinning (i.e., solid effect and cross effect), particularly towards paramagnetic metal ion polarizing agents. In this regard, novel bis-Gd(III) metal complexes have been developed for cross effect DNP as well. Furthermore, ubiquitin has been introduced as a model protein by site-directed spin labeling using various Gd(III)-binding chelate labels. This has enabled the first DNP transfer between a bound metal ion and 13C within a protein. Experiments on spin-labeled mutants as well as deuterated proteins have identified the methyl-induced dipolar relaxation between 1H and 13C as impeding factor for the build-up of nuclear spin polarization. Preliminary experiments on direct 15N polarization are nevertheless highly promising and show enhancement factors >100 as well as indications of selective enhancement of signals arising from site chains within a certain distance from the electron spin.During the extension period three currently unsolved challenges of the project will be addressed in order to conclusively answer the respective questions (Which mechanisms have to be considered for high-field DNP with metal ions? What is the distance dependence of the direct DNP transfer process? Is it possible to obtain structural information directly from DNP parameters?). Towards one aspect, bis-Mn(II) complexes will be investigated and the interplay between nuclear Larmor frequency, zero-field splitting, and hyperfine interaction to the metal nucleus will be elucidated. This information is essential for the utilization of biologically relevant Mn(II) as DNP polarizing agent, for example, towards in-cell spectroscopy. The second aspect is the investigation of direct 15N polarization on Gd(III)-labeled ubiquitin. Due to the very small gyromagnetic ratio of 15N as well as the sparse network of nitrogen atoms in comparison to carbon in proteins, homonuclear spin-diffusion is greatly attenuated. This situation is expected to conserve the site-specificity of DNP. Lastly, DNA double helical model systems will be utilized for analysis of the distance dependence of DNP and paramagnetic relaxation enhancement (PRE) under DNP conditions. For this, one strand will be labeled with an electron spin while the complementary strand will be modified with a 13C,15N-isotope label. This information (i.e., distance dependence of DNP and PRE) is invaluable in the scientific community because no quantitative model exists at the current time due to the lack of convincing experimental indications and unfeasibility of theoretical modeling under DNP-relevant conditions.

在最初的Emmy Noether项目中，可以研究魔法旋转（即固体效应和交叉效应）下的动态核极化（DNP）的相关机制，尤其是针对顺磁金属离子离子极化剂。在这方面，新型的BIS-GD（III）金属配合物也为交叉效应DNP开发了。此外，使用各种GD（III）结合的螯合物标记将泛素作为模型蛋白引入了模型蛋白。这使蛋白质内结合金属离子和13C之间的第一个DNP转移。在自旋标记的突变体以及氘代蛋白上进行的实验已经鉴定出甲基诱导的1H和13C之间的偶极弛豫是积累核自旋极化的阻碍因素。但是，直接15N极化的初步实验仍然很有希望，并且显示出增强因子> 100，以及在与电子旋转一定距离内产生的位置链产生的信号的选择性增强的指示。为了最终回答各自的问题（必须考虑使用金属离子的高场DNP的哪些机制？直接DNP传输过程的距离依赖性是什么？是否有可能直接从DNP参数获得结构信息？ ）。在一个方面，将研究BIS-MN（II）复合物，并将阐明核LARMOR频率，零场分裂和与金属核的超精细相互作用之间的相互作用。该信息对于将生物学相关的MN（II）作为DNP偏振剂（例如，朝向细胞光谱法）的利用至关重要。第二个方面是对GD（III）标记的泛素的直接15N极化的研究。与蛋白质中的碳相比，由于速磁比非常小，氮原子的稀疏网络大大减弱。预计这种情况将保留DNP的场地特异性。最后，DNA双螺旋模型系统将用于分析DNP条件下DNP和顺磁性松弛增强（PRE）的距离依赖性。为此，一条线将用电子自旋标记，而互补链将用13C，15n同位素标签进行修饰。在科学界，这些信息（即DNP和PRE的距离依赖性）是无价的，因为由于缺乏令人信服的实验指示和在DNP相关条件下理论建模的不可接受性，目前没有定量模型。