Investigation of site-specific dynamic nuclear polarization on biomolecules

在最初的Emmy Noether项目中，有可能研究魔角旋转（即固体效应和交叉效应）下动态核极化（DNP）的相关机制，特别是顺磁性金属离子极化剂。在这方面，新的双- gd （III）金属配合物也被开发用于交叉效应DNP。此外，通过使用各种Gd（III）结合螯合物标记的位点定向自旋标记，泛素已被引入作为模型蛋白。这使得结合金属离子和蛋白质中的13C之间的DNP首次转移成为可能。对自旋标记突变体和氘化蛋白的实验发现，甲基诱导的1H和13C之间的偶极弛豫是核自旋极化形成的阻碍因素。然而，直接15N极化的初步实验是非常有希望的，并显示了增强因子bbb100，以及在距离电子自旋一定距离内的位点链产生的信号有选择性增强的迹象。在延长期间，将解决项目目前尚未解决的三个挑战，以便最终回答各自的问题(金属离子高场DNP必须考虑哪些机制？直接DNP传递过程的距离依赖关系是什么？是否有可能直接从DNP参数中获得结构信息？一方面，将研究双-锰（II）配合物，并阐明核拉莫尔频率、零场分裂和与金属核的超精细相互作用之间的相互作用。这一信息对于利用生物学相关的Mn（II）作为DNP极化剂，例如，用于细胞内光谱学是必不可少的。第二个方面是Gd（III）标记的泛素上15N直接极化的研究。由于15N的陀螺磁比非常小，并且与蛋白质中的碳原子相比，氮原子的网络稀疏，因此同核自旋扩散大大减弱。这种情况有望保留DNP的部位特异性。最后，利用DNA双螺旋模型系统分析DNP和顺磁弛豫增强（PRE）在DNP条件下的距离依赖关系。为此，一条链将被标记为电子自旋，而互补链将被修饰为13C， 15n同位素标记。这些信息（即DNP和PRE的距离依赖关系）在科学界是无价的，因为目前由于缺乏令人信服的实验适应症和DNP相关条件下理论建模的不可行性，没有定量模型存在。

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.