Development of Biomolecular High Field Pulsed DNP and cwDNP MAS-NMR Applications

连续波(CW)DNP已被证明为固体核磁共振提供了前所未有的信号增强。充分挖掘其在生物分子应用方面的潜力才刚刚开始。不幸的是，它固有的逆场依赖性限制了它在磁场中的应用，与通常用于生物分子核磁共振的磁场相比，磁场更低。相比之下，脉冲DNP对信号的增强不依赖于磁场，需要进一步的研究和开发。我们建议建立一个工作在263 GHz/400 MHz的脉冲DNP固体核磁共振实验来推动这一发展。配备了微波开关的回旋管，可以产生相干的微波脉冲，将被集成到我们现有的核磁共振基础设施中。研制了一种适用于宏观有序样品的微波谐振腔固体核磁共振双共振探头。通过这种硬件设置，我们旨在评估不同的脉冲DNP机制，并将它们应用于定向和超薄膜蛋白质样品的固态核磁共振实验。我们预计，这项工作不仅将促进有序样品的使用，而且还将为我们提供有价值的数据，以便长期建立脉冲DNP MAS-核磁共振实验。在这一开发项目的同时，我们将在冷冻RNA和蛋白质溶液、膜蛋白复合体和完整的核糖体颗粒上进行cwDNP MAS-NMR应用。通过实施这一研究计划的开放用户概念，将允许访问这一独特的研究基础设施。

Continuous wave (cw)DNP has been shown to offer unprecedented signal enhancement for solidstate NMR. Exploiting its full potential for biomolecular applications is just at the beginning. Unfortunately, its intrinsic inverse field dependence limits its use to magnetic fields, which are lower, compared to those usually used for biomolecular NMR. In contrast, signal enhancement by pulsed DNP does not depend on the magnetic field but requires further research and development. We propose to establish a pulsed DNP solid-state NMR experiment operating at 263 GHz / 400 MHz to drive such development. A gyrotron equipped with microwave switches, which allows creating coherent microwave pulses, will be integrated into our existing NMR infrastructure. A solid-state NMR double resonance probe with microwave resonator for macroscopically ordered samples will be developed. With this hardware setup, we aim to evaluate different pulsed DNP mechanisms and will apply them to solid-state NMR experiments on oriented and ultra-thin membrane protein samples. We expect that this work will not only allow advancement in the use of ordered samples but also provides us with valuable data based on which pulsed DNP MAS-NMR experiments can be established in the long term. In parallel to this developmental project, we will carry out cwDNP MAS-NMR applications on frozen RNA and protein solutions, membrane protein complexes and complete ribosome particles. Access to this unique research infrastructure will be granted by implementing an open user concept for this research program.