Extended Data Fig. 3: NANOG diffusion properties.

a, fSEC and UV-SEC MW estimation of NANOG. (Top panels), SEC MW calibration plots for fSEC (h6f-NANOG WT/W8A-eGFP) and UV-SEC (MBP-NANOG WT/W8A and h6g-NANOG/Skp complex) experiments. (Bottom panel), Summary table of back-calculated MW for various proteins based on the MW calibration standards above. b, Plot of ln (R_h, hydrodynamic radii) vs ln (number of residues). The simulated lines were derived using empirical equations³⁵ (see Supplementary Table 1; black, denatured proteins; red, folded proteins). For convenience, the right and top axis labels correspond to actual R_h values and number of residues. Also plotted are actual FCS-derived R_h data (from diffusion coefficients using Stokes-Einstein equation; see Supplementary Table 1) for various NANOG constructs, SOX2 and h6g-eGFP under different experimental conditions (see Supplementary Table 1). SOX2-AF488, h6g-eGFP and W8A mutants (AF488- and GFP-tagged) fall within the boundaries for folded and denatured protein sizes. However, NANOG WT R_h data (except for data taken immediately after refolding) are significantly larger than predicted from denatured proteins. c, Photon counting histogram simulation and fitting. (Left panel), PCH simulation of two particles with different molecular brightness. Increase in molecular brightness (ε) of particles results in wider Poisson distribution. (Right panel), PCH data of h6g-eGFP (▲) and W8A mutant (■) follow a Poisson distribution and the PCH curve fits (one uniform species; blue, red, respectively) approximate the actual data. In contrast, h6f-NANOG WT-eGFP PCH full data (●) deviate significantly from a fit for one uniform species (▬, black). Segmental fitting of PCH data points results in different degrees of molecular brightness (yellow, 1ε; green ~4ε; cyan,~9ε). e, Burst analysis in mammalian cell lysates. (Left panel), Histogram depicts the number of events vs the ‘monomer’ brightness/ multiples of average counts per sec of the WT vs W8A mutant (h6f-NANOG WT/W8A-eGFP, ~20 nM). (Right panel), Histogram show the number of events vs monomer brightness of WT (NANOG-mCherry, ~20 nM) vs ΔWR mutant (NANOG ΔWR-mCherry, ~20 nM). The cells were treated with benzonase to remove protein-DNA interactions. Data shown represent 2 independent experiments.

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