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

描述（由申请人提供）：现有的分子结构测定方法，主要基于x射线晶体学和核磁共振（NMR）溶液方法，在对生物功能至关重要的不溶性蛋白质上取得了有限的成功。最近的各种发展已经增强了固体核磁共振方法的有效性，其中包括魔角样品旋转（MAS），并且这些技术还在继续取得相当大的进展。然而，事实仍然是，静止（非mas）高功率方法，如PISEMA，迄今为止在产生大型、复杂的螺旋膜蛋白结构方面更有成果。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业上可用的探针进行，只能在少数实验室中在中间磁场磁铁上的自制探针上勉强实施。在固体核磁共振方法测定大分子结构方面，一些世界上最负盛名和最成功的研究人员表示，需要大幅增加射频场强，因为在三共振1H/13C/15N探针中，需要显着提高光谱分辨率，同时显着减少射频样品加热。该第二阶段提案寻求资金，以完成超高功率三共振探头的开发，用于高达1ghz的领域，射频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：射频场强度、频谱分辨率和信噪比，每一个都有两倍以上的改进。预计最终结果将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，该方法基于500 MHz的原型5毫米探头和900 MHz的模拟。第二阶段，4毫米，900 MHz探针预计将证明：(1)能够在三个共振同时产生110 kHz以上的持续旋转框架频率，(2)静态光谱分辨率低于0.02 ppm, (3) 15N时的S/N优于70 <L天然丰度甲酰胺的50:1。实现所需的射频场强将需要4 kW的15N （91 MHz）射频脉冲，1500 W的13C （226 MHz）射频脉冲和350 W的1H （900 MHz）射频脉冲。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）中兼容。提出的工作建立在早期减少射频样品加热和提高MAS探针的功率处理和分辨率的工作基础上；它还加入了专有的新技术来实现破纪录的动力处理。第二阶段900兆赫三共振PISEMA探测器的初步现场测试预计将于第一年年底前在佛罗里达州的国家高磁场实验室进行。

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.