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），并且在不久的将来，这些技术可能会取得相当大的进展。然而，事实仍然是，静止（非mas）高功率方法迄今为止在产生大型，复杂的螺旋膜蛋白结构方面更富有成果。此外，十多年来，主要的核磁共振探头制造商在所需的探头硬件方面没有取得重大进展。在固体核磁共振方法测定大分子结构方面，几位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度，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。该提案寻求资金，开始开发一种超高功率三共振900 MHz核磁共振探针，其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、光谱分辨率和信噪比）中的每一个都有两倍以上的改进。预计最终结果将是在生物大分子的许多应用中信号采集时间减少一个数量级。第一阶段将使用500 MHz的核磁共振实验和900 MHz的工作台实验相结合来证明该方法的可行性。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持续的140 kHz以上的旋转框架频率，(2)静态光谱分辨率低于0.03 ppm,(3)在天然丰度为70¿L的甲酰胺上15N的S/N优于50:1。实现所需的射频场强度将需要4 kW的15N （90 MHz）射频脉冲，1500 W的13C （225 MHz）射频脉冲和400 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（至少1.0 GHz）中兼容。提出的工作建立在早期的工作，减少射频样品加热和提高功率处理和分辨率在MAS探针；它还添加了专有的、新颖的技术来实现破纪录的动力处理。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。

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.