An Ultra-High-Power H/C/N NMR Probe for Membrane Proteins

描述（由申请方提供）：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方面取得了有限的成功。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），并在这些技术中继续取得相当大的额外进展。然而，事实仍然是固定（非MAS）高功率方法，如PISEMA，迄今为止在产生大的，复杂的，螺旋膜蛋白的结构方面更富有成效。几个领先的研究小组最近发表的初步工作已经证明了先进的3D方法的价值，这些方法不能使用任何商用探头进行，只能在少数实验室的中场磁体中在自制探头上少量实施。在固体NMR方法测定大分子结构方面，世界上最负盛名和最成功的几位研究人员已经表示需要大幅增加RF场强，这是显著提高光谱分辨率所需的，沿着显著减少RF样品加热，在三重共振1H/13 C/15 N探针中。 该第二阶段提案寻求资金，以完成超高功率三重谐振探头的开发，其场高达1 GHz，RF样品加热的数量级减少，其余三个最重要和技术要求最高的规格中的每一个都同时提高了两倍以上：RF场强，光谱分辨率和S/N。预计最终结果是生物大分子中许多技术的信号采集时间减少一个数量级。第一阶段的工作证明了该方法的可行性的基础上，原型5毫米探头为500 MHz和模拟在900 MHz。阶段II，4 mm，900 MHz探头预期将证明以下内容：（1）能够在三个共振下同时产生高于110 kHz的持续双稳态帧频率，（2）静态光谱分辨率低于0.02 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RF场强将需要4 kW RF脉冲（15 N（91 MHz））、1500 W RF脉冲（13 C（226 MHz））和350 W RF脉冲（1H（900 MHz））。 该方法将与窄孔（NB）磁体在未来十年预计的最高磁场（1.0 GHz）下的操作兼容。拟议的工作建立在早期工作的基础上，减少RF样品加热，提高MAS探针的功率处理和分辨率;它增加了专有的新技术，以实现破纪录的功率处理。第二阶段900兆赫三重共振PISEMA探头的初步现场测试预计在第一年年底前在佛罗里达的国家高磁场实验室进行。 公共卫生相关性：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5,000多个高场NMR系统，目前NMR设备的年销售额约为3亿美元。拟议的超高功率NMR探针的发展，预计将提高能力，以确定大的，不溶性的，膜蛋白的分子结构，通过先进的NMR方法在许多情况下由一个数量级。

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 biological 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 continues. Yet, the fact remains that stationary (non-MAS) high-power methods, such as PISEMA, have been more fruitful thus far in yielding structures of large, complex, helical membrane proteins. Preliminary work recently published by several leading research groups has demonstrated the value of advanced 3D methods that cannot be carried out using any commercially available probes, and can only be marginally implemented on home-built probes in mid-field magnets in a few laboratories. 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 Phase II proposal seeks funding to complete the development of an ultra-high-power triple-resonance probe for fields up to 1 GHz 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 techniques in biological macromolecules. The Phase I effort demonstrated the feasibility of the approach based on a prototype 5-mm probe for 500 MHz and simulations 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 110 kHz at the three resonances simultaneously, (2) static spectral resolution below 0.02 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 (91 MHz), 1500 W RF pulses for 13C (226 MHz), and 350 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 - to 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 of the Phase II 900 MHz triple-resonance PISEMA probe is expected before the end of the first year at the National High Magnetic Field Laboratory in Florida. PUBLIC HEALTH RELEVANCE: 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 5,000 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 an order of magnitude in many cases.