Fig. 3: Kinetic analysis of FAD binding coupled to dCRY folding.
a–c Density plots of degree of folding of combined data at [FAD] = 0, 0.3 nM and 10 nM, respectively. The combined density plots reveal five distinct clusters (labeled C0 thru C4) with degree of folding centered at 0, 0.26 ± 0.01, 0.52 ± 0.01, 0.80 ± 0.01 and 1.0 ± 0.01, respectively (Supplementary Table 1). d–f Plots of bootstrapped refolding data as a function of time at each FAD concentration. The data were globally fitted following the mechanism shown in the scheme in (g). g Model of dCRY folding. h Density plot of combined FAD titration data displays the same five cluster centers as in the kinetic refolding data shown in (a–c). i FAD titration plot of clustered and bootstrapped data from FAD titration experiments. The solid lines represent a global fitting of the scheme in (g). Fitted parameters are listed in Supplementary Table 2. Density plots in (a–c, h) provide a probability density estimate for each data using kernel density estimation with a Gaussian kernel and the optimum bandwidth41. The y-axis of (d–f, i) labeled Fractions of dCRY states correspond to the populations of each of the clusters normalized to 1. Data in (d–f, i) are presented as mean values ± SD. For each FAD concentration at each refolding time (d–f), 6 different single molecules (i.e., biologically independent samples) for a total of 2926 data points. For (i), N = 6 biologically independent samples per FAD concentration (6 different single molecules investigated), for a total of 3039 data points. Source data of (d–f, i) are available as a Source Data file.
