Project Summary – GM132997 This proposal is focused on the development of high frequency dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) to enhance sensitivity in NMR based structural biology experiments. 1 CW DNP at 527 GHz/800 MHz: The 800/89 magnet with a sweep coil and console is operating. We plan to use the 800 MHz/527 GHz system to examine amyloid proteins and other interesting systems and to develop the methodology to perform DNP experiments at high fields. This is the sole 527 GHz/800 MHz DNP instrument available for biophysics experiments in North America. 2. Applications to Peptides and Proteins: The familial mutants of A which lead to early onset AD, are sparsely available. Thus, and structural studies require methods with high sensitivity and we intend to determine the structure of the Arctic mutant, E22G-A1-42, and other familial mutants, such as D23N. 2 High Resolution 17O NMR: With new isotope labeling procedures, high fields, and high spinning frequencies which improve sensitivity, it is finally possible to record high resolution spectra of 17O. We plan to incorporate DNP in 17O experiments to increase sensitivity and to examine model systems GGVVIA to develop the methodology and to 17O label proteins for structural studies. 3 Time Domain DNP Experiments: To translate pulsed DNP to 250 GHz we have developed a series of new swept frequency and pulsed DNP experiments. Using a fast AWG, and a TWT amplifier operating at 250 GHz, we will investigate off resonance NOVEL, the frequency sweep integrated solid effect (FS-ISE) and the stretched solid effect (SSE) and new versions of time optimized DNP (TOP DNP) as approaches for time domain polarization transfer. These should eventually replace standard CW techniques like the solid effect and cross effect. 6 1H Detected DNP: We propose to develop methods for 1H detected DNP of 13C, 15N and 17O resonances using recently developed diamond rotors and a helium recirculation system that will permit r/2>100 kHz at 90 K. The system will initially use 1.3 and 0.7 mm ZrO2 rotors and we will convert to laser machined diamond rotors which we have successfully produced that will spin at >100 kHz. The recirculation system will also be useful for spinning at ambient temperatures at >200 kHz and will permit design of a variety of new pulse experiments for structural studies.