500 - MHZ REDOR NMR SPECTROMETER

Washington University, Saint Louis MO

Investigators

Abstract

We are using new solid-state rotational-echo double resonance (REDOR) NMR experiments to characterize the structures of biologically and biomedically important macromolecular systems. These systems are not suited to analysis by diffraction or solution-state NMR methods. They are also beyond the current scope of the total-structure, solid-state NMR methods being developed by Opella, Griffin, Tycko, and others. We are developing a complementary NMR approach in which we combine available structural information about the systems or their subunits (from crystallography, solution-state NMR, electron diffraction, ab initio and homology-based calculations), with detailed REDOR information about restricted regions (interfaces, channels, binding sites) important to biological function. Currently there are 5 major users of the high-sensitivity, high-resolution 500-MHz REDOR spectrometer in the structural biology program at Washington University, and another 9 minor participants. The most severe limitation on progress in implementing our REDOR program is the available machine time. There is sufficient demand for machine time by the major users to occupy completely the present spectrometer for more than 18 months with existing samples and planned experiments. The average wait for access is 6 months. Two of the minor users have been waiting for a year for access and need time to conduct feasibility tests and collect preliminary data. Our solution to this dilemma is to create a new solid-state NMR laboratory with the help of an NIH shared-instrumentation grant and substantial support from Washington University. This facility will contain three 500-MHz REDOR spectrometers: the present machine and two clones. Adding two new spectrometers is first, cost effective (because of attractive prices for buying in bulk), and second, necessary to meet scheduling demands of both major and minor users. NIH funds will be used for one of the new spectrometers, and Washington University funds for the other. Although there is no suitable REDOR spectrometer sold by a single vendor, all of the components for the two new spectrometers are commercially available with the exception of the special REDOR probes. For these, we propose to build in-house, two 6- frequency probes with our proven (and patented), high-performance transmission-line probe technology. These probes will differ in the type of magic-angle spinning rotor employed. Thus, rotor volume and spinning speed can be tailored to the demands of specific experiments for enhanced sensitivity. We anticipate that this combination of new spectrometers and probes will multiply the productivity of our structural biology solid-state NMR laboratory by a factor of four and thereby eliminate the need for a queue for either major or minor users.