High Field DNP and EPR in Biological Systems

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Project Summary -- EB002026 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: We plan to complete development of a 527 GHz/800 MHz DNP spectrometer. It will involve (1) completion of low temperature (LT), 3 channel MAS probes, (2) the cryogenics for MAS at 80 K, (3) corrugated waveguide, and (4) interfacing these components with a 527 GHz tunable gyrotron. This will be the sole 527 GHz/800 MHz DNP instrument available in North America. 2. Applications to Peptides and Proteins: We plan to continue application of DNP to studies of proteins The specific aim is to optimize the polarizing agents and NMR methodology for 2H,13C,15N labeled amyloids, in particular tau, and to determine the structure of tau fibrils and PET imaging binding sites. We will also determine the structure of H217O \ in fibrils with 17O DNP experiments on TTR and GNNQQNY 2 High B0 Cross Effect (CE) Polarizing Agents: We plan to develop new polarizing agents for CE DNP at 600-800 MHz. They will use larger e--e- dipole couplings, trityl-TEMPO biradicals, and narrow line radicals with different isotropic g-values that satisfy the cross effect matching condition. 3 Overhauser Effect (OE) DNP in Solids: We plan to develop narrow line trityl radicals with large 1H hyperfine couplings that mediate OE DNP. The temperature, ω1S and ω0S dependence of these radicals will be studied. In addition we will study specifically 2H labeled BDPA to determine the mechanism of the OE. 4 Pulsed DNP Experiments: The specific aim is to move pulsed DNP to 250 GHz using an EPR spectrometer that is centered around a fast AWG, a 600-1000 watt 250 GHz gyroamplifier and a MAS probe with a TE011 resonator and 1.3 mm rotor. We will investigate NOVEL, off resonance NOVEL, the frequency sweep integrated solid effect (FS-ISE), and time optimized DNP (TOP DNP) as approaches for time domain polarization transfer. We plan to develop adiabatic versions of the latter. 6 1H Detected DNP: We propose to develop methods for 1H detected DNP of 13C, 15N and 17O resonances using a helium recirculation system that will permit ωr/2π>100 kHz at 90 K. The system will initially use 1.3 and 0.7 rotors from ZrO2 and we will develop laser machined diamond rotors that will spin at >100 kHz. The recirculation system will also be useful for spinning at ambient temperatures at >200 kHz.