Real-Time In-Cell Nuclear Magnetic Resonance: Ribosome-Targeted Antibiotics Modulate Quinary Protein Interactions.

How ribosome antibiotics affect a wide range of biochemical pathways is not well understood; changes in RNA-mediated protein quinary interactions and consequent activity inside the crowded cytosol may provide one possible mechanism. We developed real-time (RT) in-cell nuclear magnetic resonance (NMR) spectroscopy to monitor temporal changes in protein quinary structure, for ≥24 h, in response to external and internal stimuli. RT in-cell NMR consists of a bioreactor containing gel-encapsulated cells inside a 5 mm NMR tube, a gravity siphon for continuous exchange of medium, and a horizontal drip irrigation system to supply nutrients to the cells during the experiment. We showed that adding antibiotics that bind to the small ribosomal subunit results in more extensive quinary interactions between thioredoxin and mRNA. The results substantiate the idea that RNA-mediated modulation of quinary protein interactions may provide the physical basis for ribosome inhibition and other regulatory pathways.