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Abstract
Grant Number: 1R43RR018423-01 PI Name: DOTY, DAVID F. PI Email: david@dotynmr.com PI Title: PRESIDENT Project Title: A SAS NMR Probe for Structure Elucidation of Proteins Abstract: DESCRIPTION (provided by applicant): Available methods for 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 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 bicelles (owing to their magnetic anisotropy), but this does not permit the needed dynamical control over the amount and direction of the partial alignment. Very recent analysis and experiments by several foremost NMR research groups indicate novel Switched Angle Spinning (SAS) techniques should provide the needed dynamic control over the bicelle (and hence, the protein) alignment. When a sample containing discoidal bicelles of negative magnetic anisotropy is spun at the Magic Angle (54.7/0 with respect to B/0), their interaction with Bo vanishes and their orientation becomes random. For sample spinning at angles less than 54.7/0, 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. The instrumental requirements of SAS NMR suitable for protein structure determination are extremely challenging. The NMR probe must be capable of multinuclear triple-resonance magic angle spinning (MAS) with highly sensitive indirect (1H) detection with high resolution (~0.01 ppm) at fields up to 19 T. In addition, rapid (<30 ms) reorientation of the spinning axis is required without adversely affecting spinning stability or rf tuning on any channel; and there are a number of additional requirements, including the application of pulsed field gradients, stable temperature control, minimization of 1H background signals, and compatibility with narrow-bore high-field magnets. We have previously implemented SAS in wide-bore magnets with some of the above features at low fields. Preliminary design evaluation suggests the requirements of the high-field SAS probe can be realized within the constraints of the narrow bore magnet. This Phase I SBIR project is expected to demonstrate feasibility of a high-performance 1H/X/Y PFG-HR-SAS probe suitable for a narrow-bore magnet at 600 MHz. The prototype probe will be tested by Dr. Ad Bax at the NIH. The Phase II will complete the developments necessary for commercial products compatible with Bruker and Varian narrow-bore spectrometers at fields at least up to 800 MHz.
Thesaurus Terms:
biomedical equipment development, nuclear magnetic resonance spectroscopy, protein structure, structural biology
macromolecule
bioengineering /biomedical engineering
Institution: DOTY SCIENTIFIC, INC. 700 CLEMSON RD COLUMBIA, SC 29229 Fiscal Year: 2003 Department: Project Start: 15-MAY-2003 Project End: 14-FEB-2004 ICD: NATIONAL CENTER FOR RESEARCH RESOURCES IRG: ZRG1