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We investigate complexes of two paramagnetic metal ions Gd³⁺ and Mn²⁺ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of ¹H, ¹³C, and ¹⁵N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the m[subscript S]= -1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as ¹³C and even ¹⁵N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of > 100 (for ¹H) and on the order of 1000 (for ¹³C) can be achieved in the saturation limit even at 80 K. At larger Gd(iii) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd³⁺ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between ¹³C and ¹⁵N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd³⁺ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology., National Institutes of Health (U.S.) (Grant EB-002026), National Institutes of Health (U.S.) (Grant EB-00280)

The impact of Nuclear Magnetic Resonance (NMR) on studies of large macromolecular complexes hinges on improvements in sensitivity and resolution. Dynamic nuclear polarization (DNP) in the solid state can offer improved sensitivity, provided sample preparation is optimized to preserve spectral resolution. For a few nanomoles of intact ribosomes and an 800kDa ribosomal complex we demonstrate that the combination of DNP and magic-angle spinning NMR (MAS-NMR) allows one to overcome current sensitivity limitations so that homo- and heteronuclear 13C and 15N NMR correlation spectra can be recorded. Ribosome particles, directly pelleted and frozen into an NMR rotor, yield DNP signal enhancements on the order of ~25-fold and spectra that exhibit narrow linewidths, suitable for obtaining site-specific information. We anticipate that the same approach is applicable to other high molecular weight complexes

Dynamic nuclear polarization (DNP) has the potential to enhance the sensitivity of magic-angle spinning (MAS) NMR by many orders of magnitude and therefore to revolutionize atomic resolution structural analysis. Currently, the most widely used approach to DNP for studies of chemical, material, and biological systems involves the cross-effect (CE) mechanism, which relies on biradicals as polarizing agents. However, at high magnetic fields (≥5 T), the best biradicals used for CE MAS-DNP are still far from optimal, primarily because of the nuclear depolarization effects they induce. In the presence of bisnitroxide biradicals, magic-angle rotation results in a reverse CE that can deplete the initial proton Boltzmann polarization by more than a factor of 2. In this paper we show that these depolarization losses can be avoided by using a polarizing agent composed of a narrow-line trityl radical tethered to a broad-line TEMPO. Consequently, we show that a biocompatible trityl-nitroxide biradical, TEMTriPol-1, provides the highest MAS NMR sensitivity at ≥10 T, and its relative efficiency increases with the magnetic field strength. We use numerical simulations to explain the absence of depolarization for TEMTriPol-1 and its high efficiency, paving the way for the next generation of polarizing agents for DNP. We demonstrate the superior sensitivity enhancement using TEMTriPol-1 by recording the first solid-state 2D¹³ C- ¹³ C correlation spectrum at natural isotopic abundance at a magnetic field of 18.8 T.

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a â-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C- and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. Keyword: [PSI+] prion; solid-state NMR; amyloid; Sup35; dynamic nuclear polarization, National Institutes of Health (U.S.) (Grants GM-025874), National Institutes of Health (U.S.) (Grants EB-003151), National Institutes of Health (U.S.) (Grants EB-002804), National Institutes of Health (U.S.) (Grants EB-002026)

We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology.

Magic angle spinning (MAS) is used in solid-state NMR to remove the broadening effects of the chemical shift anisotropy (CSA). In this work we investigate a technique that can reintroduce the CSA in order to selectively invert transverse magnetization. The technique involves an amplitude sweep of the radio frequency field through a multiple of the spinning frequency. The selectivity of this inversion mechanism is determined by the size of the CSA. We develop a theoretical framework to describe this process and demonstrate the CSA selective inversion with numerical simulations and experimental data. We combine this approach with cross-polarization (CP) for potential applications in multi-dimensional MAS NMR., National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-003151)

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP., National Institutes of Health (U.S.) (Grant EB002804), National Institutes of Health (U.S.) (Grant EB003151), National Institutes of Health (U.S.) (Grant EB002026)

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008., Vita., Includes bibliographical references., he development of solid-state NMR techniques and application to amyloid fibrils are presented. In addition, a new method of selective inversion based on chemical shift anisotropy is presented. An improved method for highly accurate distance measurement across parallel [beta]-sheets in amyloid fibrils has been developed. This method combines the a double quantum filtered version of the dipolar recoupling sequence DRAWS with the simulation and data fitting program SPINEVOLUTION to provide a simple and effective way to measure interstrand distances. This method was applied to TTR105.115 fibrils. Several other methods were applied to the TTR fibrils to measure interstrand and intersheet distances including REDOR, TEDOR, R2TRW, and DARR. The intermolecular distances were combined with the previously solved monomer structure to generate a high resolution (RMSD of 1.0 Å) of the TTR protofiliment. A disease associated mutant (L 11 M) of these fibrils was also studied by solid-state NMR to determine the monomer structure. Distance measurements on this system were done via 3D TEDOR and R2W, and torsion angle were also measured. A high resolution structure of the monomer is presented. Some non-fibril related research concerning the exploration of chemical shift anisotropy and cross polarization is presented in the later chapters. Both experimental evidence and a theoretical framework are presented to demonstrate the phenomenon of selective inversion based on the size of the chemical shift anisotropy. Further work concerning the use of cross polarization selectively was developed and is presented., by Marc Anthony Caporini., Ph.D.