Investigation of site-specific dynamic nuclear polarization on biomolecules

在最初的艾美奖Noether计划中，有可能研究魔角旋转下动态核极化(DNP)的相关机制(即固体效应和交叉效应)，特别是对顺磁性金属离子极化剂的机制。在这方面，新型的双Gd(III)金属配合物也被开发出来用于交叉效应DNP。此外，泛素已经被引入为一种模型蛋白质，通过使用各种与Gd(III)结合的螯合物标记进行定点自旋标记。这使得结合的金属离子和蛋白质中的13C之间的第一次DNP转移成为可能。对自旋标记突变体和氚蛋白的实验证明，甲基诱导的1H和13C之间的偶极弛豫是核自旋极化建立的阻碍因素。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&GT；100以及从距离电子自旋的特定距离内的位置链产生的信号选择性增强的迹象。在延伸期内，将解决该项目目前尚未解决的三个挑战，以便最终回答各自的问题(对于带有金属离子的高场DNP，必须考虑哪些机制？直接DNP转移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？)一方面，我们将研究双锰(II)配合物，阐明核Larmor频率、零场分裂和超精细相互作用与金属核之间的相互作用。这一信息对于生物相关的Mn(II)作为DNP偏振剂的利用是必不可少的，例如，用于细胞内光谱。第二个方面是对Gd(III)标记泛素的直接15N极化的研究。由于15N的旋磁比非常小，以及与蛋白质中的碳相比，氮原子的稀疏网络，同核自旋扩散大大减弱。这种情况有望保持DNP的位置特异性。最后，利用DNA双螺旋模型系统分析了DNP的距离依赖性以及DNP条件下的顺磁弛豫增强效应(PRE)。为此，一条链将被标记有电子自旋，而互补链将被标记13C，15N-同位素标记。这一信息(即DNP和Pre的距离相关性)在科学界是非常宝贵的，因为由于缺乏令人信服的实验迹象，以及在DNP相关条件下理论建模的不可行性，目前还不存在定量模型。

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.