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

描述(申请人提供)：现有的分子结构确定方法，主要基于X射线结晶学和核磁共振(核磁共振)溶液方法，在对生物功能至关重要的不溶蛋白质方面取得的成功有限。最近的各种发展提高了结合魔角样品旋转(MAS)的固体核磁共振方法的有效性，这类技术还在继续取得相当大的进展。然而，事实仍然是，固定(非MAS)高功率方法，如PISEMA，到目前为止在产生大的、复杂的螺旋膜蛋白的结构方面取得了更多的成果。几个领先的研究小组最近发表的初步工作证明了先进3D方法的价值，这些方法无法使用任何商业探测器进行，只能在少数实验室的中场磁铁的自制探测器上实现。几位世界上最负盛名和最成功的固体核磁共振方法确定大分子结构的研究人员表示，需要大幅增加射频场强，以显著提高光谱分辨率，同时大幅降低1H/13C/15N三谐振探头的RF样品加热。这项第二阶段的提案寻求资金，以完成对高达1 GHz场强的超高功率三谐振探头的开发，使射频样品加热减少数量级，其余三个最重要和技术要求最苛刻的规范中的每一个同时提高两倍以上：射频场强、光谱分辨率和S/N。预计最终结果将是生物大分子中的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，该方法基于500 MHz的5 mm探头原型和900 MHz的模拟。第二阶段，4 mm，900 MHz探头预计将展示以下内容：(1)能够在三个共振下同时产生110 kHz以上的持续旋转框架频率，(2)静态光谱分辨率低于0.02ppm，以及(3)S/N在15N处优于50：1在70&lt；L天然丰度甲酰胺。要达到所需的射频场强，15N(91 MHz)需要4 kW的RF脉冲，13C(226 MHz)需要1500 W的RF脉冲，1H(900 MHz)需要350 W的RF脉冲。该方法将与窄孔(Nb)磁体在未来十年预期的最高磁场-1.0 GHz下的操作兼容。这项拟议的工作建立在减少射频样品加热和提高MAS探头的功率处理和分辨率的早期工作的基础上；它还增加了专利和新技术，以实现创纪录的功率处理。第二阶段900兆赫三谐振PISEMA探头的初步现场测试预计将在第一年年底之前在佛罗里达州的国家高磁场实验室进行。 与公共卫生相关：在未来十年，人们对确定人体内15,000种膜蛋白的结构有着强烈的医学和科学兴趣，尽管现有的核磁共振和X射线方法效果不佳，在过去十年中只产生了几种这样的结构。全球安装了5000多台高场核磁共振系统，目前核磁共振设备的年销售额约为3亿美元。所提出的超高功率核磁共振探针的开发有望在许多情况下提高利用先进的核磁共振方法确定大的、不可溶的膜蛋白的分子结构的能力。

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.