Resonator Approach to Pulsed Dynamic Nuclear Polarization of Membrane Proteins

膜蛋白脉冲动态核极化的谐振器方法

• 批准号: 10242008
• 负责人: Alexander A. Nevzorov
• 金额: $ 25.85万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-18 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242008
• 关键词: Aluminum Oxide; Amplifiers; Benchmarking; Biological; Biological Process; Biomedical Research; Biomedical Technology; Capsid Proteins; Cells; Ceramics; Computer Simulation; Crystallization; Data; Detection; Development; Diamond; Disease; Drug Targeting; Electron Spin Resonance Spectroscopy; Electrons; Environment; Exhibits; Freezing; Frequencies; Gramicidin; Heating; High temperature of physical object; Hydration status; Label; Lipid Bilayers; Lipids; Magnetism; Measures; Membrane; Membrane Proteins; Metals; Methodology; Methods; Modeling; Modernization; Molecular; Nanoporous; Nature; Nuclear; Nuclear Magnetic Resonance; Pattern; Peptides; Phase; Physiologic pulse; Physiological; Positioning Attribute; Radiation; Relaxation; Research Personnel; Risk; Sampling; Scheme; Series; Signal Transduction; Source; Spin Labels; Structure; Surface; Techniques; Technology; Temperature; Testing; Time; Water; absorption; attenuation; base; cryogenics; design; design and construction; experience; experimental study; high risk; improved; innovation; instrument; instrumentation; interest; macromolecule; nanopore; novel; photonics; prototype; radio frequency; reconstitution; solid state; solid state nuclear magnetic resonance; structural biology; success; tool

Nuclear Magnetic Resonance (NMR) is an exceptionally versatile and informative spectroscopic technique for atomic-level structure-function studies of biological macromolecules in their native-like environments. In particular, solid-state NMR allows one to study membrane proteins in lipid bilayers under the conditions approaching those encountered in the biological cells. Membrane proteins are of particular interest for biomedicine being implicated in numerous biological processes and diseases and constituting nearly 50% of the modern drug targets. However, low polarization of the nuclear spins limits NMR sensitivity and represents the major roadblock for expanding its use in structural biology. Dynamic nuclear polarization (DNP) can potentially boost sensitivity of NMR by up to several hundred times via irradiating the sample with mm-waves at matching frequencies. Despite significant progress, DNP NMR of biological samples above the freezing temperatures remains to be a challenge mainly because of short relaxation times of the nuclear and electron spins at higher temperatures and excessive sample heating by mm-waves. We propose to overcome these fundamental problems by constructing a novel 200 GHz/300 MHz DNP spectrometer which will be based on resonant mm-wave structures and will operate in a pulse mode for DNP transfer vs. the continuous mode currently in use. The key innovation is our recently invented mm-wave photonic band-gap resonators which increase the sample volume by approximately 1-2 orders of magnitude as compared to the existing resonator cavity designs. We propose to increase the quality factors of such resonators from Q=200 as demonstrated for the prototype to at least Q=1,000 in order to boost mm-wave field at the sample. Achieving these higher mm- wave fields will be essential for enabling advanced pulse schemes for DNP that will provide maximum NMR signal enhancements while minimizing sample heating. The spectrometer development will be guided by computer simulations of mm-wave fields and pulse DNP sequences, and will be based on the existing low- power prototype operating in a continuous DNP mode yielding record-breaking preliminary data obtained at room temperature. The spectrometer will operate over a broad temperature range (100-330 K), and multi- resonance probeheads will be optimized for hydrated biological samples above the freezing point. The new DNP technology will be applied to a series of biological samples including hydrated membrane proteins aligned by nanoporous substrates. Success of the project will be built upon the extensive expertise of the two collaborating PIs (Nevzorov and Smirnov) in designing and constructing a room temperature DNP NMR spectrometer prototype based on solid-state mm-wave components. The new pulsed DNP spectrometer will open up unexplored perspectives with regard to developing novel pulse methodologies for DNP-enhanced solid-state NMR of membrane proteins. This is a high-gain high-risk project where the risk is leveraged by the extensive experience of the investigators and the highly encouraging preliminary results. 1

核磁共振（NMR）是一种非常通用和信息丰富的光谱技术，用于 生物大分子在类天然环境中的原子水平结构-功能研究。在 特别是，固态NMR允许人们研究脂质双层中的膜蛋白， 接近生物细胞中遇到的那些。膜蛋白是特别感兴趣的， 生物医学与许多生物过程和疾病有关，占全球生物医学的近50%。 现代药物靶点然而，核自旋的低极化限制了NMR灵敏度，并且代表了 这是在结构生物学中扩大其应用的主要障碍。动态核极化（DNP）可以 通过用毫米波照射样品，可能将NMR的灵敏度提高数百倍 频率匹配。尽管取得了重大进展，但生物样品在冰点以上的DNP NMR 温度仍然是一个挑战，主要是因为核和电子的弛豫时间短， 在更高的温度下旋转和由毫米波引起的过度样品加热。我们建议克服这些困难， 基本问题，通过构建一种新型的200 GHz/300 MHz DNP光谱仪，将基于 谐振毫米波结构，并将在脉冲模式下工作，用于DNP传输与连续模式 目前正在使用。关键的创新是我们最近发明的毫米波光子带隙谐振器， 与现有的谐振器相比，将样品体积增加大约1-2个数量级 型腔设计我们建议从Q=200增加这种谐振器的品质因数，如对于 将原型至少提高到Q=1,000，以增强样品处的毫米波场。实现这些更高的mm- 波场将是必不可少的，使先进的脉冲方案的DNP，将提供最大的NMR 信号增强，同时最大限度地减少样品加热。光谱仪的开发将遵循以下原则： 毫米波场和脉冲DNP序列的计算机模拟，并将基于现有的低， 动力原型在连续DNP模式下运行，获得了破纪录的初步数据， 室温光谱仪将在宽温度范围（100-330 K）和多个温度范围内工作。 共振探头将针对高于凝固点的水合生物样品进行优化。新 DNP技术将应用于一系列生物样品，包括水化膜蛋白对齐 通过纳米多孔基质。该项目的成功将建立在两人广泛的专业知识之上 合作PI（Nevzorov和Smirnov）设计和构建室温DNP NMR 基于固态毫米波元件的光谱仪原型。新的脉冲DNP光谱仪将 开辟了未开发的前景，关于发展新的脉冲方法，为DNP增强 膜蛋白的固态NMR。这是一个高收益高风险的项目，风险是由 调查人员的丰富经验和非常令人鼓舞的初步结果。 1