Dynamic Nuclear Polarization of Deuterated Proteins

For D's a jolly good fellow: Deuteration of proteins significantly increases the signal enhancements observed in dynamic nuclear polarization (DNP) magic-angle spinning (MAS) NMR experiments. In 13C CP-MAS spectra an enhancement of 120 is observed for perdeuterated SH3 with an exchangeable proton ratio of 50 %, whereas the enhancement is only 31 for the fully protonated SH3. The direct 13C excitation of the perdeuterated sample increases the enhancement to 148

DNP Enhanced Frequency-Selective TEDOR Experiments in Bacteriorhodopsin

We describe a new approach to multiple [superscript 13]C–[superscript 15]N distance measurements in uniformly labeled solids, frequency-selective (FS) TEDOR. The method shares features with FS-REDOR and ZF- and BASE-TEDOR, which also provide quantitative [superscript 15]N–[superscript 13]C spectral assignments and distance measurements in U-[[superscript 13]C,[superscript 15]N] samples. To demonstrate the validity of the FS-TEDOR sequence, we measured distances in [U-[superscript 13]C,15N]-asparagine which are in good agreement with other methods. In addition, we integrate high frequency dynamic nuclear polarization (DNP) into the experimental protocol and use FS-TEDOR to record a resolved correlation spectrum of the Arg-[superscript 13]Cγ–[superscript 15]Nε region in [U-[superscript 13]C,15N]-bacteriorhodopsin. We resolve six of the seven cross-peaks expected based on the primary sequence of this membrane protein.National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-001960)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-002804)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-001035)National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant Number EB-002026

Resolution and Polarization Distribution in Cryogenic DNP/MAS Experiments

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz [superscript 1]H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between [superscript 1]H–[superscript 1]H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026)National Institutes of Health (U.S.) (Grant EB001965)National Institutes of Health (U.S.) (Grant EB004866)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Cryogenic sample exchange NMR probe for magic angle spinning dynamic nuclear polarization

We describe a cryogenic sample exchange system that dramatically improves the efficiency of magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments by reducing the time required to change samples and by improving long-term instrument stability. Changing samples in conventional cryogenic MAS DNP/NMR experiments involves warming the probe to room temperature, detaching all cryogenic, RF, and microwave connections, removing the probe from the magnet, replacing the sample, and reversing all the previous steps, with the entire cycle requiring a few hours. The sample exchange system described here—which relies on an eject pipe attached to the front of the MAS stator and a vacuum jacketed dewar with a bellowed hole—circumvents these procedures. To demonstrate the excellent sensitivity, resolution, and stability achieved with this quadruple resonance sample exchange probe, we have performed high precision distance measurements on the active site of the membrane protein bacteriorhodopsin. We also include a spectrum of the tripeptide N-f-MLF-OH at 100 K which shows 30 Hz linewidths.National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002804)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-001960)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-001035)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-003151)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Solid-State Dynamic Nuclear Polarization at 263 GHz: Spectrometer Design and Experimental Results

Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz 1H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water–glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.National Institutes of Health (U.S.) (NIH grant EB-002804)National Institutes of Health (U.S.) (NIH grant EB-002026

Dynamic Nuclear Polarization NMR Spectroscopy Allows High-Throughput Characterization of Microporous Organic Polymers

Dynamic nuclear polarization (DNP) solid-state NMR was used to obtain natural abundance 13C and 15N CP MAS NMR spectra of microporous organic polymers with excellent signal-to-noise ratio, allowing for unprecedented details in the molecular structure to be determined for these complex polymer networks. Sensitivity enhancements larger than 10 were obtained with bis-nitroxide radical at 14.1 T and low temperature (∼105 K). This DNP MAS NMR approach allows efficient, high-throughput characterization of libraries of porous polymers prepared by combinatorial chemistry methods