MRI: Development of a pulsed ESR/NMR DNP spectrometer for structural studies of membrane proteins in native lipid environments

将开发一种脉冲DNP/NMR/ESR （DyNE）光谱仪，用于研究膜蛋白在天然生理条件下的宏观排列结构。固态核磁共振（ssNMR）允许人们在不需要结晶的情况下获得膜蛋白和膜结合受体在其天然脂质环境中的原子分辨率结构。目前ssNMR的缺点是由于核自旋在可用磁场下的极化较小，灵敏度相对较低。这一主要缺陷将通过采用和优化动态核极化（DNP）方法来解决，该方法将较大的极化从电子自旋转移到原子核，从而实现数百倍的核磁共振信号增强。DNP正在迅速革新小分子、聚合物和无机表面的核磁共振领域，但其在生物大分子中的应用仍然相当有限。该项目的重点是开发DNP仪器，该仪器将特别适合于膜蛋白在其生理相关条件下的结构-功能研究（即37℃下的完全水合作用）。为了实现这些目标，PI （Nevzorov）在ssNMR膜蛋白高分辨率结构研究方面的专业知识将与Co-PI （Smirnov）的专业知识相结合。Smirnov是使用脂质纳米管阵列稳定膜蛋白的先驱，也是构建毫米波仪器和电子自旋共振探针（ESR）的专家。DyNE仪器的开发将在磁共振、膜蛋白生物物理学和纳米技术的界面上进行。这种仪器将成为从生物物理学家到结构生物学家和药物化学家等不同研究人员研究小分子治疗药物与膜蛋白受体相互作用的宝贵工具。在这个开发项目的过程中，研究生将获得跨学科的培训，从先进的核磁共振和ESR方法到膜蛋白的生物物理实验和混合纳米材料的制备。该仪器开发的成果将通过跨学科教育计划进行传播，包括NCSU现有的本科和研究生课程，以及与生物学家、化学家和物理学家合作，利用这种独特的仪器进行膜蛋白的结构研究。

A pulsed DNP/NMR/ESR (DyNE) spectrometer for the structural studies of macroscopically aligned membrane proteins under their native-like physiological conditions will be developed. Solid-state nuclear magnetic resonance (ssNMR) allows one to obtain atomic-resolution structures of membrane proteins and membrane-bound receptors in their native lipid environment without the need of crystallization. The current disadvantage of ssNMR is its relatively low sensitivity due to small polarization of the nuclear spins at the available magnetic fields. This main drawback will be addressed by employing and optimizing the Dynamic Nuclear Polarization (DNP) method that transfers the larger polarization from the electronic spins to the nuclei, thereby achieving several hundred-fold enhancements of the NMR signals. DNP is rapidly revolutionizing the field of NMR of small molecules, polymers, and inorganic surfaces, but its applications to biological macromolecules remain fairly limited. This project is focused on developing the DNP instrumentation that will be specifically suited for the structure-function studies of membrane proteins under their physiologically relevant conditions (i.e., full hydration at 37 C). To achieve these goals, the expertise of the PI (Nevzorov) in high-resolution structural studies of membrane proteins by ssNMR will be combined with the expertise of the Co-PI (Smirnov), who is a pioneer of using lipid nanotube arrays for stabilizing membrane proteins and an expert in building millimeter-wave instrumentation and probeheads for electron-spin resonance (ESR). The development of the DyNE instrument will take place at the interface of magnetic resonance, membrane protein biophysics, and nanotechnology. Such an instrument would become an invaluable tool for a diverse group of researchers ranging from biophysicists to structural biologists and medicinal chemists studying the interactions of small-molecule therapeutics with membrane protein receptors. In the course of this development project graduate students will obtain an interdisciplinary training ranging from advanced NMR and ESR methods to biophysical experiments with membrane proteins and preparation of hybrid nanomaterials. The results from this instrumentation development will be disseminated through interdisciplinary educational initiatives including the existing undergraduate and graduate level courses at NCSU as well as by collaborating with biologists, chemists, and physicists on the structural studies of membrane proteins by utilizing this unique instrumentation.