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：
10004143
负责人：
Alexander A. Nevzorov
金额：
$ 35.68万
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 2022-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10004143
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) 是一种用途广泛、信息丰富的光谱技术，可用于生物大分子在其天然环境中的原子级结构功能研究。在特别是，固态核磁共振允许人们在一定条件下研究脂质双层中的膜蛋白接近生物细胞中遇到的那些。膜蛋白特别令人感兴趣生物医学与许多生物过程和疾病有关，占生物医学的近 50% 现代药物靶标。然而，核自旋的低极化限制了 NMR 灵敏度并代表扩大其在结构生物学中的应用的主要障碍。动态核极化 (DNP) 可以通过用毫米波照射样品，可将 NMR 的灵敏度提高数百倍在匹配的频率。尽管取得了重大进展，生物样品的 DNP NMR 仍高于冰点温度仍然是一个挑战，主要是因为核和电子的弛豫时间很短在较高温度下旋转并通过毫米波过度加热样品。我们建议克服这些通过构建新型 200 GHz/300 MHz DNP 光谱仪来解决基本问题，该光谱仪将基于谐振毫米波结构，将在 DNP 传输的脉冲模式下运行，而不是在连续模式下运行目前正在使用。关键的创新是我们最近发明的毫米波光子带隙谐振器，它与现有谐振器相比，样品体积增加约1-2个数量级腔体设计。我们建议从 Q=200 开始增加此类谐振器的品质因数，如下所示原型至少 Q=1,000，以增强样品处的毫米波场。实现这些更高的目标波场对于实现 DNP 的先进脉冲方案至关重要，该方案将提供最大的 NMR 信号增强，同时最大限度地减少样品加热。光谱仪的发展将遵循毫米波场和脉冲 DNP 序列的计算机模拟，并将基于现有的低在连续 DNP 模式下运行的动力原型产生破纪录的初步数据室温。该光谱仪可在较宽的温度范围（100-330 K）和多共振探头将针对冰点以上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，包括水合膜蛋白对齐通过纳米多孔基材。该项目的成功将建立在两人广泛的专业知识的基础上与 PI（Nevzorov 和 Smirnov）合作设计和构建室温 DNP NMR 基于固态毫米波组件的光谱仪原型。新型脉冲 DNP 光谱仪将在开发 DNP 增强型新型脉冲方法方面开辟了未探索的前景膜蛋白的固态核磁共振。这是一个高收益高风险的项目，风险由研究人员的丰富经验和非常令人鼓舞的初步结果。 1