DNP-Assisted NMR Investigation of Proteins at Endogenous Levels in Cellular Milieu

In-Cell Sensitivity-Enhanced NMR of Intact Living Mammalian Cells

“…Here, we eliminated carbon and nitrogen-containing components of the freezing media because doing so will facilitate spectral interpretation for samples where the target molecule is at concentrations that are low enough that signals from natural abundance components make a significant contribution to the spectra. 27 However, DNP efficiency is unlikely to be compromised by nutrient-rich components of the freezing media since cell lysates and other complex mixtures of biomolecules support efficient DNP. 1,27,37 Thus, as long as samples are frozen slowly and cryogenically transferred into a pre-cooled spectrometer 40 , the freezing media can be tailored to the cell line.…”

“…[21][22][23][24][25][26] With the sensitivity gains conferred by dynamic nuclear polarization (DNP), MAS NMR has the sensitivity to detect proteins at their endogenous concentrations in complex biological environments. 1, 21,22,27 However, the effectiveness of DNP-enhanced MAS NMR is critically dependent on sample composition [28][29][30][31][32][33][34] and experimental conditions 23,[35][36][37][38] . DNP increases the sensitivity of NMR spectroscopy through the transfer of the large spin polarization of an unpaired electron to nearby nuclei 35 which are typically introduced into a sample by doping with millimolar concentrations of stable biological radicals [32][33][34] .…”

Targeted DNP for biomolecular solid-state NMR

“…More details can be found in the recent review from the Frederick group. 108 DNP experiments need to be performed relatively rapidly after addition of the PA in order to limit radical reduction. 109 In this approach, the challenge is to detect the labeled protein at μM concentration, which can be overcome using multiple-quantum filtered pulse sequences that only select NMR-active nuclei that are close in space.…”

Targetable Tetrazine‐Based Dynamic Nuclear Polarization Agents for Biological Systems

“…With now routine signal enhancements in the 100‐fold range, DNP has aided the in vitro structural studies of amyloid polymers, membrane proteins, and biological material such as collagen, bones and tissues . Improvements in DNP instrumentation, sample preparation, and polarization agent design have also allowed NMR spectroscopists to turn their attention to the cellular environment, where the structural analysis of endogenous concentrations of biological macromolecules has come within reach . In a typical DNP experiment, the sample is doped with millimolar amounts of polarization agents, small molecules that contain two stable unpaired‐electron spins.…”