19F NMR strategy for probing site-specific RNA dynamics in large RNA-protein complexes: application to the Hfq RNA chaperone

• RNA binding proteins alter RNA motions to chaperone regulatory interactions. • Site-specific uridine labeling with 5-19F,6-2H yields high quality NMR spectra. • Uridines on different surfaces of Hfq experience different conformational dynamics. • 19F labeling and NMR can reveal the dynamics of large RNA-protein complexes. Nuclear magnetic resonance (NMR) spectroscopy is uniquely suited to probing motions on the microsecond to millisecond timescales that underlie RNA recognition and restructuring of ribonucleoprotein (RNP) complexes. However, the large size and conformational dynamics of most RNPs results in poor sensitivity, spectral crowding, and resolution loss in traditional NMR approaches. Here, we present a 19 F NMR strategy for resolving residue-level RNA conformational dynamics within a ∼76 kDa Hfq-RNA complex at micromolar concentrations. Site-specific incorporation of dual-labeled 6- 2 H, 5- 19 F-uridine (6D-5FU) into a 27-nt model RNA yields excellent spectral quality when in complex with the Hfq chaperone. 19 F resonance broadening and 19 F Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments reveal specific changes in RNA dynamics on different surfaces of Hfq. Our NMR strategy provides a framework for studying how RNA-binding proteins like Hfq selectively alter the conformational dynamics of their RNA targets.