Extended Data Fig. 6: Determination of bimolecular association rate of V2Rpp peptide and four distinct β-arrestin sensors from pre- steady state PRE- smFRET measurements.
From: Parallel stopped-flow interrogation of diverse biological systems at the single-molecule scale
(a) Observed binding rates, kobs, were determined at concentrations from 2 to 10 µM of V2Rpp from the observed rate of entry into the low FRET (active) state following peptide addition by rapid computer-controlled injection. Dissociation rates, \({k}_{\rm{off}}^{\rm{app}}\), were estimated from rate of return to stable high FRET (basal) state following wash out of V2Rpp. Bimolecular association rate constant, \({k}_{\rm{on}}^{\rm{app}}\), was determined by fitting of the concentration dependance of kobs. Error bars in observed binding rates denote SEM determined from 1000 bootstrap resamples. (b) Measured bimolecular association rates from pre-steady-state PRE-smFRET measurements of V2Rpp induced β-arrestin1 conformational change, determined by linear fitting of (a). Error bars denote associated standard error of slope from linear fitting. P values are from F-test.
