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High-resolution structural studies of proteins and protein complexes in a native eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins in intact cells. Most exogenous delivery techniques are limited to specific cell types or are too destructive to preserve cellular physiology. The feasibility of microfluidics transfection or volume exchange for convective transfer, VECT, as a means to deliver labeled target proteins to HeLa cells for in-cell NMR experiments is demonstrated. VECT delivery does not require optimization or impede cell viability; cells are immediately available for long-term eukaryotic in-cell NMR experiments. In-cell NMR-based drug screening using VECT was demonstrated by collecting spectra of the sensor molecule DARPP32, in response to exogenous administration of Forskolin., (© 2022. The Author(s).)

Protein-protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real time in-cell NMR spectroscopy, RT-NMR, utilizes a bioreactor to maintain cells in an active metabolic state. Improvement in bioreactor technology maintains ATP levels at >95% for up to 24 hours, enabling protein overexpression and a previously undetected interaction between prokaryotic ubiquitin-like protein, Pup, and mycobacterial proteasomal ATPase, Mpa, to be detected. Singular value decomposition, SVD, of the NMR spectra collected over the course of Mpa overexpression easily identified the PPIs despite the large variation in background signals due to the highly active metabolome.