DNP enhanced solid-state NMR of lattice-like microcrystalline protein assemblies facilitated by co-assembly with dinitroxide-tagged proteins.

Dynamic nuclear polarization (DNP) enables dramatic sensitivity enhancements of solid-state nuclear magnetic resonance (NMR) spectra for biological systems to be realized via polarization transfer from unpaired electrons of a polarizing agent, typically a nitroxide biradical, to nuclear spins of the system of interest. Here, we systematically investigate the prospect of recording DNP enhanced solid-state NMR spectra of hydrated lattice-like protein assemblies within sample formulations that are free of the commonly employed glycerol-based glassy solvent matrix containing exogenous polarizing agent molecules and instead consist of isotope-enriched protein of interest co-assembled with its dinitroxide-tagged structural analog incorporated as a dopant at low concentration. As a model system we use microcrystals of 13C,15N-labeled immunoglobulin-binding domain of protein G (GB1) doped with different amounts in the ∼1-16 % regime of a GB1 variant, 28R1/32R1, containing nitroxide spin labels at residues 28 and 32. For 28R1/32R1 dopant concentration of 4 %, equivalent to ca. 5 mM polarizing agent in the microcrystals, the DNP signal enhancement for 13C,15N-GB1 was found to be comparable to that obtained for model amino acid 13C,15N-proline in the usual glycerol/D2O/H2O matrix containing 5 mM 28R1/32R1 as the polarizing agent; further sensitivity increases were observed for 28R1/32R1 concentrations on the order of 10 %. Moreover, the spectral resolution for hydrated 13C,15N-GB1 microcrystals doped with 28R1/32R1 at ∼100 K was similar to that for a microcrystal suspension in glycerol/D2O/H2O with AMUPol biradical. This sample formulation approach based on doping with dinitroxide-tagged proteins is expected to be applicable to not only protein microcrystals but also to other lattice-like or filamentous assemblies composed of small to medium-size protein subunits, and further major improvements in sensitivity can be anticipated for this approach with continued development of optimal covalent nitroxide biradical DNP polarizing agent tags.