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

将开发一台脉冲DNP/核磁共振/电子自旋共振(DYNE)光谱仪，用于研究宏观排列的膜蛋白在天然生理条件下的结构。固体核磁共振技术可以在天然的脂质环境中获得膜蛋白和膜结合受体的原子分辨结构，而不需要结晶。目前的缺点是核磁共振的灵敏度相对较低，这是因为在可用的磁场下，核自旋的极化很小。这一主要缺点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正在给小分子、聚合物和无机表面的核磁共振领域带来革命性的变化，但它在生物大分子中的应用仍然相当有限。这个项目的重点是开发DNP仪器，该仪器将特别适合于在与生理相关的条件下(即37℃完全水合)膜蛋白的结构-功能研究。为了实现这些目标，PI(Nevzorov)在单核磁共振高分辨率膜蛋白结构研究方面的专业知识将与Co-Pi(Smirnov)的专业知识相结合，Co-PI(Smirnov)是使用脂质纳米管阵列稳定膜蛋白的先驱，也是建造毫米波仪器和电子自旋共振(ESR)探头的专家。Dyne仪器的开发将在磁共振、膜蛋白质生物物理和纳米技术的交界处进行。这样的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各种研究人员研究小分子疗法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，范围从先进的核磁共振和电子自旋共振方法到膜蛋白的生物物理实验和杂化纳米材料的制备。这一仪器开发的结果将通过跨学科的教育举措传播，包括NCSU现有的本科生和研究生水平的课程，以及通过利用这种独特的仪器与生物学家、化学家和物理学家合作研究膜蛋白的结构。

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.