Administrative Supplement: Biophysical investigations of RNA complexes essential for gene expression

项目总结/文摘

PROJECT SUMMARY/ABSTRACT This administrative supplement proposal is for a custom-built 800 MHz solid state NMR (SSNMR) probe that will allow detection of 1H, 19F, 31P, 13C and 15N nuclei. Until now, such probes have only been available for wide bore magnets at a maximum frequency of 600 MHz. Now, Phoenix NMR has developed a standard bore magic-angle spinning probe with a 1.6 mm rotor capable of spinning up to 40 kHz, with the full range of tuning configurations to enable molecular structure determination at 800 MHz. The radio frequency solenoid coil can be tuned simultaneously to three or four frequencies, including all the triple resonance combinations required for standard applications (HPC, HCN, HPN) as well as novel, quadruple resonance configurations involving 19F, such as HFPC, HFCN, and HFPN. The Phoenix probe is the only standard bore design available that supports 1H decoupling while observing 19F and two other nuclei. We anticipate the combined improvements in resolution and sensitivity at 800 MHz will enable complete assignments of biomolecules and complexes of molecular weight 20 to 50 kDa, as well as studies of dynamics utilizing dipolar order parameter and relaxation methods. The probe will be used for projects in the Butcher and Henzler-Wildman laboratories and will be placed in the National Magnetic Resonance Facility at Madison (NMRFAM) at UW-Madison, which has recently recruited SSNMR expert Prof. Chad Rienstra (formerly U. Illinois). Thus, the probe will support at least 3 groups from within UW- Madison and will also be accessible to an extensive outside user base. The Butcher lab will use the probe to investigate poly-UG RNA and its association with the protein TDP-43. Due to the highly repetitive RNA sequence, these studies will make use of site-specific 2′ fluoro groups on the RNA. A 1.0 Å crystal structure of the free RNA has been solved and NMR assignments have been made, which will facilitate structure determination of the complex by SSNMR. The Henzler-Wildman lab will use the probe to study how the bacterial EmrE multi-drug transporter and its interaction with fluorinated substrates. HN-resolved 1H-19F REDOR experiments have been performed that demonstrate the feasibility of the project.