Supplementary Figure 2: In vitro characterization of FLINC.

(A) Size-exclusion chromatography permits a qualitative assessment of the interaction between Dronpa and TagRFP-T. Sizes of the protein oligomers are as indicated. A peak at 92 kDa was observed only in the mixed solution, indicating a trace amount of a 3-fluorescent-protein hetero-oligomer formed at protein concentrations of 60 μM each. Color codes: red, TagRFP-T; green, Dronpa; orange, algebraic sum of Dronpa/TagRFP-T chromatographs; blue, mixture. (B) SedAnal plot of ΔC as a function of cell radius for fitting of Dronpa+TagRFP-T mixed sample data from analytical ultracentrifugation. The experimental data are shown as black dots, while the calculated fitting curve for the A + B ↔ C model is shown as a solid blue line. Data from scans 55 to 100 are included in this fit; the input parameters include: S(Dronpa) = 2.15, S(TagRFP-T) = 3.30, and S(Dronpa+TagRFP-T) = 4.0. See Supplementary Information. (C) Absorption and (D) fluorescence spectroscopy of purified DpTT fractions compared to TagRFP-T or Dronpa. (E) Electrostatic surfaces of Dronpa and TagRFP-T calculated by the Adaptive Poisson-Boltzmann Solver (APBS) plug-in in PyMOL. The basic pocket in Dronpa and the acidic patch around the chromophore in TagRFP-T, both features believed to be important for FLINC, have been highlighted.