AN ULTRA-HIGH-POWER H/C/N NMR PROBE FOR MEMBRANE PROTEINS

用于膜蛋白的超高功率 H/C/N NMR 探针

• 批准号: 7220199
• 负责人: Francis DAVID Doty
• 金额: $ 17.46万
• 依托单位: DOTY SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7220199
• 关键词: A-factor (Streptomyces); Biological; Compatible; Complex; Crystallography; Development; Effectiveness; Equipment; Faculty; Frequencies; Funding; Future; Goals; Heating; Human body; Institution; Lanthanoid Series Elements; Magic; Manufacturer Name; Medical; Membrane; Membrane Proteins; Methods; Molecular Structure; NMR Spectroscopy; Noise; Nuclear Magnetic Resonance; Operative Surgical Procedures; Performance; Phase; Physiologic pulse; Proteins; Pulse taking; Range; Research Personnel; Resolution; Roentgen Rays; Sales; Sampling; Signal Transduction; Solid; Solutions; Solvents; Structure; System; Techniques; Temperature; Testing; Time; Voice; Width; Work; base; cost; design; desire; formamide; improved; interest; macromolecule; new technology; prototype; quantum; research study; success

DESCRIPTION (provided by applicant): Available methods for molecular structure determination, based primarily on x-ray crystallography and Nuclear Magnetic Resonance (NMR) solution methods, have had limited success on the insoluble proteins that are critical to bio- logical function. Various recent developments have enhanced the effectiveness of solids NMR methods incorporating Magic Angle sample Spinning (MAS), and considerable additional progress in such techniques appears likely in the near future. Yet, the fact remains that stationary (non-MAS) high-power methods have been more fruitful thus far in yielding structures of large, complex, helical membrane proteins. Moreover, the major NMR probe manufacturers have not offered significant advances in the needed probe hardware in more than a decade. Several of the world's most prestigious and successful researchers in macromolecule structure determination by solids NMR methods have voiced the need for major increases in rf field strength, as required for significantly improved spectral resolution, along with dramatically reduced rf sample heating, in triple-resonance 1H/13C/15N probes. This proposal seeks funding to begin the development of an ultra-high-power triple-resonance 900 MHz NMR probe with order-of-magnitude reduction in rf sample heating and more than a factor of two improvement in each of the remaining three most important and technically demanding specifications simultaneously: rf field strength, spectral resolution, and S/N. The net result is expected to be an order of magnitude reduction in signal acquisition time for many applications in biological macromolecules. The Phase I will demonstrate the feasibility of the approach using a combination of NMR experiments at 500 MHz and work-bench experiments at 900 MHz. The Phase II, 4 mm, 900 MHz probe is expected to demonstrate the following: (1) ability to generate sustained rotating-frame frequencies above 140 kHz at the three resonances simultaneously, (2) static spectral resolution below 0.03 ppm, and (3) S/N on 15N better than 50:1 on 70 ¿L of natural-abundance formamide. Achieving the desired rf field strengths will require 4 kW rf pulses for 15N (90 MHz), 1500 W rf pulses for 13C (225 MHz), and 400 W rf pulses for 1H (900 MHz). The approach will be compatible with operation in narrow-bore (NB) magnets at the highest fields anticipated in the coming decade - at least 1.0 GHz. The proposed work builds on earlier work in reducing rf sample heating and improving power handling and resolution in MAS probes; and it adds proprietary, novel technologies to achieve record- shattering power handling. Initial field testing at an outside institution at 900 MHz is expected by the end of the first year in Phase II. There is strong medical and scientific interest in determining the structures of the 15,000 membrane proteins in the human body over the next decade, though available NMR and X-ray methods work poorly and have yielded only a few such structures over the past decade. There are more than 4,000 high-field NMR systems installed world-wide, and annual NMR equipment sales are currently ~$300M. The proposed ultra- high-power NMR probe development is expected to enhance the ability to determine molecular structures of large, insoluble, membrane proteins by advanced NMR methods by more than an order of magnitude in many cases.

描述（由申请人提供）：主要基于X射线晶体学和核磁共振（NMR）溶液法的分子结构测定可用方法在对生物功能至关重要的不溶性蛋白质上取得的成功有限。最近的各种发展已经提高了固体NMR方法的有效性，包括魔角样品旋转（MAS），并在不久的将来出现在这样的技术相当大的额外进展。然而，事实仍然是，固定（非MAS）高功率的方法已经更富有成效，迄今为止，在产生大的，复杂的，螺旋膜蛋白的结构。此外，主要的NMR探针制造商在十多年内没有在所需的探针硬件方面提供重大进展。世界上最负盛名和最成功的几个研究人员在高分子结构测定固体核磁共振方法已经表示，需要在射频场强度的重大增加，作为显着提高光谱分辨率，沿着显着减少射频样品加热，在三重共振1H/13 C/15 N探头所需的。该提案寻求资金，以便开始开发超高功率三重共振900 MHz NMR探头，该探头可将射频样品加热降低一个数量级，并同时提高其余三个最重要和技术要求最高的规格（射频场强、光谱分辨率和信噪比）的两倍以上。预期净结果是对于生物大分子中的许多应用，信号采集时间减少一个数量级。第一阶段将使用500 MHz的NMR实验和900 MHz的工作台实验的组合来证明该方法的可行性。Phase II，4 mm，900 MHz探头预计将证明以下几点：（1）能够在三个共振处同时产生高于140 kHz的持续扫描帧频率，（2）静态光谱分辨率低于0.03 ppm，以及（3）15 N上的S/N优于70 µ L天然丰度甲酰胺上的50：1。实现所需的射频场强度将需要4千瓦的射频脉冲为15 N（90 MHz），1500 W的射频脉冲为13 C（225 MHz），和400 W的射频脉冲为1 H（900 MHz）。该方法将与未来十年预期的最高磁场（至少1.0 GHz）下的窄孔（NB）磁体中的操作兼容。拟议的工作建立在早期工作的基础上，减少射频样品加热和提高功率处理和分辨率的MAS探头;它增加了专有的，新颖的技术，以实现创纪录的功率处理。在第二阶段的第一年年底，预计将在外部机构进行900 MHz的初步现场测试。 在未来十年中，人们对确定人体中15，000种膜蛋白的结构有着强烈的医学和科学兴趣，尽管现有的NMR和X射线方法效果不佳，并且在过去十年中只产生了少数这样的结构。全球安装了4,000多个高场NMR系统，目前NMR设备的年销售额约为3亿美元。所提出的超高功率NMR探针开发预期在许多情况下将通过先进的NMR方法确定大的不溶性膜蛋白的分子结构的能力提高超过一个数量级。