This research proposes to develop solid state Dynamic Nuclear Polarization (DNP) to achieve NMR signal enhancements of >200 at room temperature, representing a significant advancement over currently employed DNP spectrometers operating at 100 Kelvin. Solid state NMR is well suited to probe atomic level structure and molecular dynamics of membrane proteins and amyloid fibrils. However, low inherent sensitivity limits solid state NMR measurements. Sensitivity from electron paramagnetic resonance (EPR) can be transferred to NMR to boost signals by more than two orders of magnitude in a process known as DNP. These ultrasensitive DNP experiments currently require samples to be frozen to below 100 Kelvin. Performing NMR based structural biology at cryogenic temperatures has significant drawbacks including perturbing molecular structure and a loss of spectral resolution. We propose to implement DNP at room temperature with new fast frequency tuning microwave sources (gyrotrons) to enable time domain DNP. These new gyrotrons will have an irradiation bandwidth of >600 MHz compared to currently available sources of 1 MHz, resulting in much better EPR control. We will also design new microwave resonance structures to improve microwave penetration, while reducing microwave heating. The new DNP technology and methodology will be demonstrated with structural and molecular dynamic studies of activators bound to Protein Kinase C regulatory domains. Achieving NMR sensitivity gains of >200 at room temperature will greatly expand the scope and precision of NMR based structural biology.
Proteins either embedded in the membranes of cells or assembled into amyloid fibrils are the targets of many drugs to combat HIV/AIDS, cardiovascular disease, and Alzheimer's disease. This research will develop new technologies to determine the structures and motion of these drug targets in order to design new medicines that are more potent and less toxic.
|Scott, Faith J; Saliba, Edward P; Albert, Brice J et al. (2018) Frequency-agile gyrotron for electron decoupling and pulsed dynamic nuclear polarization. J Magn Reson 289:45-54|
|Albert, Brice J; Gao, Chukun; Sesti, Erika L et al. (2018) Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents. Biochemistry 57:4741-4746|
|Sesti, Erika L; Saliba, Edward P; Alaniva, Nicholas et al. (2018) Electron decoupling with cross polarization and dynamic nuclear polarization below 6?K. J Magn Reson 295:1-5|
|Scott, Faith J; Sesti, Erika L; Choi, Eric J et al. (2018) Magic angle spinning NMR with metallized rotors as cylindrical microwave resonators. Magn Reson Chem 56:831-835|
|Saliba, Edward P; Sesti, Erika L; Alaniva, Nicholas et al. (2018) Pulsed Electron Decoupling and Strategies for Time Domain Dynamic Nuclear Polarization with Magic Angle Spinning. J Phys Chem Lett 9:5539-5547|
|Chen, Pinhui; Albert, Brice J; Gao, Chukun et al. (2018) Magic angle spinning spheres. Sci Adv 4:eaau1540|
|Scott, Faith J; Alaniva, Nicholas; Golota, Natalie C et al. (2018) A versatile custom cryostat for dynamic nuclear polarization supports multiple cryogenic magic angle spinning transmission line probes. J Magn Reson 297:23-32|
|Albert, Brice J; Niu, Austin; Ramani, Rashmi et al. (2017) Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation. Sci Rep 7:7456|
|Albert, Brice J; Pahng, Seong Ho; Alaniva, Nicholas et al. (2017) Instrumentation for cryogenic magic angle spinning dynamic nuclear polarization using 90L of liquid nitrogen per day. J Magn Reson 283:71-78|
|Saliba, Edward P; Sesti, Erika L; Scott, Faith J et al. (2017) Electron Decoupling with Dynamic Nuclear Polarization in Rotating Solids. J Am Chem Soc 139:6310-6313|
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