Dynamic Nuclear Polarization with Electron Decoupling in Intact Human Cells and Cell Lysates

Abstract

Dynamic nuclear polarization (DNP) is used to improve the inherently poor sensitivity of nuclear magnetic resonance spectroscopy by transferring spin polarization from electrons to nuclei. However, DNP radicals within the sample can have detrimental effects on nuclear spins close to the polarizing agent. Chirped microwave pulses and electron decoupling (eDEC) attenuate these effects in model systems, but this approach is yet to be applied to intact cells or cellular lysates. Herein, we demonstrate for the first time exceptionally fast 1H T1DNP times of just 200 and 300 ms at 90 and 6 K, respectively, using a newly synthesized methylated trityl radical within intact human cells. We further demonstrate that eDEC can also be applied to intact human cells and human and bacterial cell lysates. We investigate eDEC efficiency at different temperatures, with different solvents, and with two trityl radical derivatives. At 90 K, eDEC yields a 13C signal intensity increase of 8% in intact human cells and 10% in human and bacterial cell lysates. At 6 K, eDEC provides larger intensity increases of 15 and 39% in intact human cells and cell lysates, respectively. Combining the manipulation of electron spins with frequency-chirped pulses and sample temperatures approaching absolute zero is a promising avenue for executing rapid, high-sensitivity magic-angle spinning DNP in complex cellular environments.

Solid-State NMR Investigations of Extracellular Matrixes and Cell Walls of Algae, Bacteria, Fungi, and Plants

“…Are we prepared for high-throughput and automatic analyses of unlabeled ECMs and carbohydrate-rich biomaterials? Could we obtain high-resolution views of the sugar components in mammalian and human cells? , How should we handle cellular mixtures like the communication and interface between microbes and plants/humans? When could we completely get rid of isotope labeling?…”

Overhauser Dynamic Nuclear Polarization with Selectively Deuterated BDPA Radicals

“…Dynamic nuclear polarization (DNP) has emerged as a powerful method to overcome the inherent low sensitivity of nuclear magnetic resonance (NMR) spectroscopy by transferring the large polarization of unpaired electron spins to the nuclei of interest, typically 1 H. − DNP provides sensitivity enhancements of 2–3 orders of magnitude, enabling the study of biomolecules and materials that are sensitivity limited − and where structural studies using dipole recoupling are difficult. − The success of these experiments has stimulated the sustained development of continuous-wave (CW) solid-state devices and gyrotrons that generate microwave powers ranging from several hundred milliwatts to tens of watts, respectively. In turn, these sources provide microwave irradiation to drive electron or electron–nuclear spin transitions that facilitate CW-DNP, enabling experiments at magnetic fields up to 21.1 T. − …”

Challenges and Advances in the Application of Dynamic Nuclear Polarization to Liquid-State NMR Spectroscopy

“…Meier et al observed acetate influx and subsequent metabolic consequences in yeast . Meier et al and Balbach et al have presented comprehensive reviews of the applications of in-cell hyperpolarized NMR. , It should be noted that solid-state in-cell DNP NMR is an emerging field. , However, only liquid-state DNP has the potential to provide information about dynamic processes such as conformational changes, reaction kinetics, and evolution of metabolite profiles in intact cells.…”

“…77,78 It should be noted that solid-state in-cell DNP NMR is an emerging field. 79,80 However, only liquid-state DNP has the potential to provide information about dynamic processes such as conformational changes, reaction kinetics, and evolution of metabolite profiles in intact cells.…”