DNP-assisted solid-state NMR enables detection of proteins at nanomolar concentrations in fully protonated cellular environments

Abstract

With the sensitivity enhancements conferred by dynamic nuclear polarization (DNP), magic angle spinning (MAS) solid state NMR spectroscopy experiments can attain the necessary sensitivity to detect very low concentrations of proteins. This potentially enables structural investigations of proteins at their endogenous levels in their biological contexts where their native stoichiometries with potential interactors is maintained. Yet, even with DNP, experiments are still sensitivity limited. Moreover, when an isotopically-enriched target protein is present at physiological levels, which typically range from low micromolar to nanomolar concentrations, the isotope content from the natural abundance isotopes in the cellular milieu can outnumber the isotope content of the target protein. Using isotopically enriched yeast prion protein, Sup35NM, diluted into natural abundance yeast lysates, we optimized sample composition we find that modest cryoprotectant concentrations and fully protonated environments support efficient DNP. We experimentally validated theoretical calculations of the limit of specificity for an isotopically enriched protein in natural abundance cellular milieu. We establish that, using pulse sequences that are selective for adjacent NMR-active nuclei, proteins can be specifically detected in cellular milieu at concentrations in the hundreds of nanomolar. Finally, we find that maintaining native stoichiometries of the protein of interest to the components of the cellular environment may be important for proteins that make specific interactions with cellular constituents.

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells

“…To determine the relative populations of the α-helical rich and the intrinsically disordered forms of α-syn present inside cells, we collected highly signal averaged 1D 13 C spectra of threonine labeled α-syn inside cells. To specifically isolate signals from these sites in 1D from the natural abundance isotopes in the cellular biomass, we used a pulse sequence that reported only on 13 C sites that are within one bond of an 15 N site (38) to increase the specificity for the labeled sites in α-syn by 200-fold (39,40). We collected 15 N-filtered 13 C spectra of HEK293 cells containing 75 µM of threonine-labeled α-syn when AMUPol was introduced either by electroporation or incubation.…”