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. ? ? ?
