We propose to acquire a state-of-the-art 800 MHz wide-bore solid-state NMR spectrometer for studies of complex biomacromolecular assemblies including prion protein amyloid fibrils and large nucleosome arrays. These systems play a major role in diverse biological phenomena such as the pathogenesis of protein conformational diseases, gene regulation and DNA repair, and are of fundamental importance to human health. Most samples employed in these studies exist natively as high-molecular weight non-crystalline or fibrous aggregates. Since such preparations are insoluble and lack long-range order they are not readily interrogated by conventional X-ray crystallography or solution-state NMR methods. On the other hand, they are ideal targets for analysis by solid-state NMR. The proposed instrument will be equipped for advanced multidimensional magic-angle spinning NMR experiments, that will enable the resolution and assignment of 1H, 13C and 15N signals throughout 13C,15N-labeled proteins within complexes having molecular masses up to ~400 kDa. The instrument will also permit determination of long-range interatomic 1H-1H, 13C-13C and 13C-15N distance restraints up to ~5-8 ?, as well as 15N-Cu2+ and 13C-Cu2+ distances up to ~20-25 ? in protein molecules modified at specific sites with paramagnetic Cu2+-chelating tags. Distance restraints of this type are a key ingredient of solid-state NMR structure determination protocols. Moreover, the instrument will provide the capability to readily detect 31P, 2H and 17O nuclei in peptides, proteins and nucleic acids for structural and dynamic measurements, with simple adjustments of the standard probe tuning elements. The novel capabilities of the proposed instrument, which are critical to the success of the proposed experiments, include significantly enhanced sensitivity and resolution relative to low- to moderate-field spectrometers, excellent long-term operational stability for 2D, 3D and 4D NMR experiments, extended operation at low temperatures, and rapid magic-angle spinning at frequencies ? 40 kHz. The proposed instrument will support ongoing projects among a core group of investigators at The Ohio State University and Case Western Reserve University in Cleveland, OH with NIH R01 funding, and also enable the application of ultrahigh-field magic-angle-spinning solid-state NMR to research projects at Ohio State which can significantly benefit from this methodology but have not exploited it as of yet due to lack of equipment. The PI has extensive training in advanced magic-angle-spinning solid-state NMR applied to biological macromolecules and has developed multidimensional NMR methods for protein assignments, three-dimensional protein structure refinement and rapid acquisition of protein NMR data with enhanced sensitivity. These and other new methodologies will be implemented on the new 800 MHz NMR instrument to enable studies of biomacromolecular systems that are difficult or impossible to investigate at lower magnetic fields.

Agency
National Institute of Health (NIH)
Institute
Office of The Director, National Institutes of Health (OD)
Type
Biomedical Research Support Shared Instrumentation Grants (S10)
Project #
1S10OD012303-01
Application #
8334722
Study Section
Special Emphasis Panel (ZRG1-BCMB-R (30))
Program Officer
Levy, Abraham
Project Start
2012-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2014-06-30
Support Year
1
Fiscal Year
2012
Total Cost
$2,000,000
Indirect Cost
Name
Ohio State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210