Figure 2: Application of ¹⁹F-NMR probes of β-arrestin-1 allowed the detection of phosphate binding and structural changes by ¹⁹F-NMR spectroscopy.

(a) Upfield shifts in the ¹⁹F-NMR spectra of F2Y-phospho-sensing-probes of β-arrestin-1 are observed in response to V2Rpp binding. The chemical shifts are referenced to trifluoroacetic acid (TFA, −76.5 ppm) as an internal standard. (b) ¹⁹F-NMR spectra of β-arrestin-1–Y21-F2Y and β-arrestin-1–Y63-F2Y after titration with V2Rpp. Left: two distinct peaks were observed for Y21 after titration with V2Rpp. The peak at −135.216 p.p.m. increased (representing the V2Rpp-bound state) as the peak at −130.181 p.p.m. (representing the state of β-arrestin-1 alone) decreased, indicating a slow conformational exchange. Right: a single peak was observed for Y63; the chemical shift varied in response to differences in the concentration of the V2Rpp, suggesting that rapid conformational changes occur at this site after V2Rpp binding. (c) Binding of V2R5p, which lacks the first three phosphates in V2Rpp (pT347, pS350 and pS357), caused an upfield shift at the K107-F2Y position but no detectable change at the Y63-F2Y position (Δp.p.m.<0.05). (d) Plots of the distance root mean square deviations (RMSDs) for individual residues between inactive β-arrestin-1 and the V2Rpp-bound β-arrestin-1 complex (upper panel) and between inactive rod arrestin and its constitutively active p44 variant (lower panel). The vertical axis shows all heavy-atom RMSDs per arrestin residue. The colour code shown in the upper right corner of the panel indicates the Cα deviation of each residue. The three loops that exhibit major conformational changes are highlighted: finger loop, red; middle loop, light blue; and lariat loop, light yellow. (e) The ¹⁹F-NMR spectra of β-arrestin-1 incorporating site-directed F2Y revealed dynamic conformational changes after activation by V2Rpp. (f). Heavy-atom RMSD versus the relative amplitude of the ¹⁹F-NMR chemical shift for specific F2Y incorporation sites.