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Dynamic nuclear polarization (DNP) of a biomolecule tagged with a polarizing agent has the potential to not only increase NMR sensitivity but also to provide specificity towards the tagging site. Although the general concept has been often discussed, the observation of true site-specific DNP and its dependence on the electron–nuclear distance has been elusive. Here, we demonstrate site-specific DNP in a uniformly isotope-labeled ubiquitin. By recombinant expression of three different ubiquitin point mutants (F4C, A28C, and G75C) post-translationally modified with a Gd(3+)-chelator tag, localized metal-ion DNP of (13)C and (15)N is investigated. Effects counteracting the site-specificity of DNP such as nuclear spin-lattice relaxation and proton-driven spin diffusion have been attenuated by perdeuteration of the protein. Particularly for (15)N, large DNP enhancement factors on the order of 100 and above as well as localized effects within side-chain resonances differently distributed over the protein are observed. By analyzing the experimental DNP built-up dynamics combined with structural modeling of Gd(3+)-tags in ubiquitin supported by paramagnetic relaxation enhancement (PRE) in solution, we provide, for the first time, quantitative information on the distance dependence of the initial DNP transfer. We show that the direct (15)N DNP transfer rate indeed linearly depends on the square of the hyperfine interaction between the electron and the nucleus following Fermi’s golden rule, however, below a certain distance cutoff paramagnetic signal bleaching may dramatically skew the correlation. Direct DNP transfer rates can be used to measure electron-nuclear distances and to provide site-specificity in NMR.

We report on the spontaneous polarization transfer from dynamically hyperpolarized 1H to 13C during magic-angle spinning dynamic nuclear polarization (DNP) at temperatures around 100 K. The transfer is mediated by 1H–13C cross-relaxation within methyl groups due to reorientation dynamics, and results in an inverted 13C NMR signal of enhanced amplitude. Further spreading of transferred polarization can then occur via 13C–13C spin-diffusion. The resulting process is equal to the nuclear Overhauser effect (NOE) where typically continuous saturation of 1H by radio frequency irradiation is employed. Here, hyperpolarization by irradiation with microwaves in the presence of typical bis-nitroxide polarizing agents is utilized for steady-state displacement of 1H polarization from thermal equilibrium and perpetual spin-lattice relaxation. An effective 13C enhancement factor of up to −15 has been measured. Presence of Gd(III) furthermore amplifies the effect likely by accelerated relaxation of 1H. We provide experim...

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., Physical Chemistry Chemical Physics, 18 (39), ISSN:1463-9084, ISSN:1463-9076