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

连续波（cw）DNP已被证明为固态NMR提供前所未有的信号增强。开发其生物分子应用的全部潜力才刚刚开始。不幸的是，其固有的逆场依赖性限制了其在磁场中的使用，与通常用于生物分子NMR的磁场相比，磁场较低。相比之下，脉冲DNP的信号增强不依赖于磁场，但需要进一步的研究和开发。我们建议建立一个在263 GHz / 400 MHz工作的脉冲DNP固态NMR实验，以推动这种发展。一个配备微波开关的回旋管，可以产生相干微波脉冲，将被集成到我们现有的NMR基础设施中。研制了一种用于宏观有序样品的微波共振固体核磁共振双共振探针。通过这种硬件设置，我们的目标是评估不同的脉冲DNP机制，并将其应用于定向和超薄膜蛋白质样品的固态NMR实验。我们希望这项工作不仅可以促进有序样品的使用，而且还为我们提供了有价值的数据，基于这些数据，可以长期建立脉冲DNP MAS-NMR实验。在这个开发项目的同时，我们将在冷冻RNA和蛋白质溶液，膜蛋白复合物和完整的核糖体颗粒上进行cwDNP MAS-NMR应用。访问这个独特的研究基础设施将通过实施一个开放的用户概念，为这个研究计划授予。

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.