High Field DNP and EPR in Biological Systems

项目摘要--EB002026 本提案的重点是发展高频动态核极化(DNP)。 在基于核磁共振的结构生物学实验中提高灵敏度的核磁共振技术。 1CW 527 GHz/800 MHz DNP：我们计划完成527 GHz/800 MHz DNP的开发 分光计。它将涉及(1)完成低温(LT)，3通道MAS探头，(2) 用于80K下MAS的低温，(3)波纹波导，以及(4)将这些组件与527接口 GHz可调谐回旋管。这将是北美唯一可用的527 GHz/800 MHz DNP仪器。 2.在多肽和蛋白质方面的应用：我们计划继续应用DNP来研究 蛋白质的具体目的是优化2H，13C，15N标记的极化剂和核磁共振方法 淀粉样蛋白，特别是tau，并确定tau纤维的结构和PET成像结合部位。我们 还将通过TTR和GNNQNY上的17O DNP实验确定纤维中H_(17)O的结构 2种高B0交叉效应(CE)偏振剂：我们计划开发用于CE的新型偏振剂 DNP在600-800 MHz。他们将使用更大的e-e-偶极耦合，三-节奏双自由基和窄线 满足交叉效应匹配条件的各向同性g值不同的根。 3固体中的Overhauser效应(OE)DNP：我们计划开发具有大1H的窄线三叔丁基自由基 调节OE DNP的超精细耦合。这些自由基的温度、ω1和ω0的依赖关系将 被研究。此外，我们还将专门研究2H标记的BDPA，以确定OE的机制。 4脉冲DNP实验：具体目标是使用EPR将脉冲DNP移动到250 GHz 以快速AWG、600-1000瓦250 GHz陀螺放大器和MAS探头为中心的光谱仪 带有TE011谐振器和1.3毫米转子。我们将调查小说，非共振小说，频率 扫描集成立体效应(FS-ISE)和时间优化DNP(TOP DNP)作为时间域方法 极化转移。我们计划开发后者的绝热版本。 61H检测DNP：我们建议建立13C、15N和17O的1H检测DNP的方法 共振使用氦气再循环系统，将允许ωr/2π&gt；100 khz在90 K。该系统将 最初使用1.3和0.7来自氧化锆的转子，我们将开发激光加工的钻石转子，旋转速度为 &gt；100 kHz。再循环系统对于在200千赫的环境温度下纺纱也是有用的。

Project Summary -- EB002026 This proposal is focused on the development of high frequency dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) to enhance sensitivity in NMR based structural biology experiments. 1 CW DNP at 527 GHz/800 MHz: We plan to complete development of a 527 GHz/800 MHz DNP spectrometer. It will involve (1) completion of low temperature (LT), 3 channel MAS probes, (2) the cryogenics for MAS at 80 K, (3) corrugated waveguide, and (4) interfacing these components with a 527 GHz tunable gyrotron. This will be the sole 527 GHz/800 MHz DNP instrument available in North America. 2. Applications to Peptides and Proteins: We plan to continue application of DNP to studies of proteins The specific aim is to optimize the polarizing agents and NMR methodology for 2H,13C,15N labeled amyloids, in particular tau, and to determine the structure of tau fibrils and PET imaging binding sites. We will also determine the structure of H217O \ in fibrils with 17O DNP experiments on TTR and GNNQQNY 2 High B0 Cross Effect (CE) Polarizing Agents: We plan to develop new polarizing agents for CE DNP at 600-800 MHz. They will use larger e--e- dipole couplings, trityl-TEMPO biradicals, and narrow line radicals with different isotropic g-values that satisfy the cross effect matching condition. 3 Overhauser Effect (OE) DNP in Solids: We plan to develop narrow line trityl radicals with large 1H hyperfine couplings that mediate OE DNP. The temperature, ω1S and ω0S dependence of these radicals will be studied. In addition we will study specifically 2H labeled BDPA to determine the mechanism of the OE. 4 Pulsed DNP Experiments: The specific aim is to move pulsed DNP to 250 GHz using an EPR spectrometer that is centered around a fast AWG, a 600-1000 watt 250 GHz gyroamplifier and a MAS probe with a TE011 resonator and 1.3 mm rotor. We will investigate NOVEL, off resonance NOVEL, the frequency sweep integrated solid effect (FS-ISE), and time optimized DNP (TOP DNP) as approaches for time domain polarization transfer. We plan to develop adiabatic versions of the latter. 6 1H Detected DNP: We propose to develop methods for 1H detected DNP of 13C, 15N and 17O resonances using a helium recirculation system that will permit ωr/2π>100 kHz at 90 K. The system will initially use 1.3 and 0.7 rotors from ZrO2 and we will develop laser machined diamond rotors that will spin at >100 kHz. The recirculation system will also be useful for spinning at ambient temperatures at >200 kHz.