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

基本信息

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
项目类别：
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

项目摘要

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）是一种非常通用且信息丰富的光谱技术原子水平的结构功能研究生物大分子在其本机样环境中。在特定的固态NMR允许在条件下研究脂质双层中的膜蛋白接近在生物细胞中遇到的人。膜蛋白特别感兴趣生物医学与许多生物学过程和疾病有关，占近50％现代药物目标。然而，核自旋的极化限制了NMR敏感性，并表示扩大其在结构生物学中使用的主要障碍。动态核极化（DNP）可以通过用MM波照射样品，可能会提高NMR的灵敏度多达几百倍在匹配频率下。尽管取得了重大进展，但冻结上方的生物样品的DNP NMR 温度仍然是一个挑战，主要是因为核和电子的松弛时间较短在较高的温度下进行旋转，并通过MM波加热过多的样品。我们建议克服这些基本问题通过构建新型的200 GHz/300 MHz DNP光谱仪，该光谱仪将基于谐振MM波结构，并将在DNP传输的脉冲模式下工作与连续模式目前正在使用。关键创新是我们最近发明的MM波光谱带谐振器，该谐振器与现有谐振器相比，将样品体积增加约1-2个数量级腔设计。我们建议将此类谐振器的质量因素从Q = 200增加，如至少Q = 1,000的原型以提高样品的MM波场。实现这些更高的MM- 波场对于为DNP启用高级脉冲方案至关重要，DNP将提供最大的NMR 信号增强，同时最大程度地减少样品加热。光谱仪的开发将由 MM波场和脉冲DNP序列的计算机模拟，将基于现有的低 - 在连续DNP模式下运行的功率原型，可获得创纪录的初步数据室温。光谱仪将在较大的温度范围内运行（100-330 K），并且共振探针将针对冰点上方的水合生物样品进行优化。新的 DNP技术将应用于一系列生物样品，包括对齐的水合膜蛋白由纳米多孔底物。该项目的成功将建立在两者的广泛专业知识上在设计和构建室温DNP NMR方面合作PIS（Nevzorov和Smirnov）光谱仪原型基于固态MM波组件。新的脉冲DNP光谱仪将关于开发DNP增强的新型脉冲方法的未开发观点膜蛋白的固态NMR。这是一个高增强的高风险项目，其中风险由调查人员的丰富经验以及极为令人鼓舞的初步结果。 1