Available methods for biological molecular structure determination, based primarily on x-ray crystallography and Nuclear Magnetic Resonance (NMR) solution methods, have limitations. While the x-ray methods are several orders of magnitude faster, NMR techniques are required to obtain dynamical information, or information on biological molecules in their native environments, both of which are essential for function elucidation. One of the more promising recent additions to the arsenal of NMR tools has been the use of long-range constraints from residual dipolar couplings in partially aligned solutions. Partial macromolecule alignment has been obtained by using dilute liquid crystal solutions of disc-shaped particles called bicelles, but this does not permit the needed dynamical control over the alignment. Very recent analyses and experiments by several foremost NMR research groups indicate novel Switched Angle Spinning (SAS) techniques should provide the needed dynamic control over the bicelle (hence, the protein) alignment. When a sample containing discoidal bicelles of negative magnetic anisotropy is spun at 54.7x with respect to B0 in Magic Angle Spinning (MAS), their interaction with B0 vanishes and their orientation becomes random. For sample spinning at angles less than 54.7x, they align with their normals perpendicular to the spinning axis, while spinning at greater angles causes their normals to align with the spinning axis. Dynamic control over the spinning axis is expected to provide the protein alignment control needed for more effective utilization of the bond angle information inherent in the residual dipolar coupling. This instrument will also facilitate NMR techniques needed for the study of insoluble proteins, and a closely related variant, an H/X/Y HR MAS with a high-performance Magic Angle Gradient, will enable Frydman's ultra-fast multidimensional NMR techniques in inhomogeneous systems. The instrumental requirements of SAS NMR suitable for protein structure determination are extremely challenging. The NMR probe must be capable of multinuclear triple-resonance MAS with highly sensitive indirect (1H) detection with high resolution (0.005 ppm) at fields up to 19 T. In addition, rapid (25 ms) reorientation of the spinning axis is required without adversely affecting spinning stability or rf tuning; and there are a number of additional requirements, including pulsed field gradients, stable temperature control, low 1H background signals, and compatibility with narrow-bore (NB) high-field magnets. The Phase I project demonstrated feasibility of a high-performance 1H/X/Y PFG-HR-SAS probe suitable for a NB magnet at 600 MHz, though a number of optimization and product refinement issues remain. The Phase II will complete these developments necessary for commercial products compatible with Bruker, JEOL, and Varian NB spectrometers at fields up to 800 MHz. The Phase II will include testing of the instruments by Dr. Ad Bax, chief scientist at NIH-NIDDK.
