Extended Data Fig. 5: Full FCS curves with logarithmic binning.
Donor and acceptor autocorrelations (green, red) and donor-acceptor crosscorrelations (purple; same color scheme as in Fig. 2h, which shows the same data and fits but on a linear scale and normalized to an amplitude of 1 at ±3 μs) of ProTαC (labeled at position 56 and 110) at 128 mM ionic strength (TEK buffer with 120 mM KCl) as an unbound monomer in solution (a), in the 1:1 complex with H1 (b), and within ProTα–H1 droplets (c). For each sample, the three correlations are fitted globally (black solid lines, see Methods) with shared correlation times for translational diffusion (τD), triplet blinking (τT), dye rotation (τrot), and conformational dynamics (τcd); photon antibunching (τab) is fitted individually. τcd was then converted to the reconfiguration time of the chain, τr, as previously described79 (we note that the conversion from τcd to τr does not entail a large change in timescale, and τcd and τr differ by less than 20% in all cases investigated here). τD, τT, τrot, τr, and τab are shown in the panels if the corresponding term was included in the fit function (Eq. 6), and they point to their respective timescales. The value of τr reported here is the mean of three measurements, as in Fig. 2h, and corresponds to the distance correlation time between the dyes at position 56 and 110.79 τT in the donor-acceptor cross correlation in (B) shows a small negative amplitude, possibly indicating a slight contribution of slower distance dynamics on the microsecond timescale. Note that the deviation between fit and measurement in (c) for the translational diffusion component is caused by sample scanning, which was required to improve statistics inside the droplets.
