Fig. 4: Multiperspective smFRET imaging of β-arrestin1 activation.

a, Activation of β-arrestin1 upon binding to V2Rpp, a phospho-peptide mimetic of the phosphorylated C tail of V2R. One labeling perspective (C-tail sensor 1) is shown. b, Overview of the generic protein immobilization strategy for smFRET microarrays using adaptor molecules. This strategy enabled PRE-smFRET imaging of β-arrestin1 activation upon V2Rpp addition from four structural perspectives: C-tail release sensors 1 and 2, finger loop conformational changes and C-domain rotation. c, Representative smFRET traces showing steady-state conformational changes in β-arrestin1 C-tail sensor 1 at increasing concentrations of V2Rpp. Oligo, oligonucleotide. d, Titration of V2Rpp against arrestin measured from four structural perspectives in parallel. Plotted data represent mean and s.d. from three independent repeats. e, A representative smFRET trace showing the activation and deactivation of β-arrestin1 C-tail sensor 1 upon V2Rpp injection and washout. Binding rates were estimated from the waiting time distribution between V2Rpp injection to first transit to the low FRET active state, t_on. Dissociation rates were estimated from the distribution of waiting times between V2Rpp washout and the adoption of the stable high FRET basal state, t_off. f, PRE-smFRET imaging of β-arrestin1 activation and deactivation upon V2Rpp injection and rinsing, correspondingly; 10 µM V2Rpp was injected at t_i = 5 s and rinsed at t_r = 25 s (vertical dashed lines). Contour plots represent data compiled from 15 injections, without post-synchronization.