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. 2021 Feb 8;12:856. doi: 10.1038/s41467-020-18911-w

Fig. 6. Assembly and disassembly of bipartite split mFAP fragments m14 and m58.

Fig. 6

a–d Assembly of split mFAP fragments. a Association model in which BCLXL is fused to m58 (BCLXL_m58) (violet cartoon), aBCLXL is fused to m14 (m14_aBCLXL) (yellow cartoon), and fluorescence of DFHBI-1T (green spheres) is activated upon association (arrow) of BCLXL_m58 and m14_aBCLXL. b Normalized fluorescence intensity (points) of BCLXL_m58 titration into a constant m14_aBCLXL concentration in excess DFHBI-1T after reaching equilibrium, showing the fit to a bimolecular association model (line) using non-linear least squares fitting. c Split mFAP competitor pre-incubation model in which fluorescence of DFHBI-1T (green spheres) is activated upon competition (arrow) of m14_aBCLXL with unfused aBCLXL (yellow cartoons) for the BCLXL-binding cleft of BCLXL_m58 (violet cartoon) (the reaction evolves analogously for BFL1–aBFL1 and BCL2–aBCL2 cognate-binding partners). d Temporal evolution of fluorescence fold-change in excess DFHBI-1T upon (n = 1) addition of equimolar m14_aBFL1 (orange points) or buffer (black points) to pre-incubated equimolar BFL1_m58 and aBFL1, addition of equimolar m14_aBCL2 (violet points) or buffer (black points) to pre-incubated equimolar BCL2_m58 and aBCL2, and addition of equimolar m14_aBCLXL (blue points) or buffer (black points) to pre-incubated equimolar BCLXL_m58 and aBCLXL, showing the fits to a monophasic exponential function (lines) using non-linear least squares fitting. e, f Disassembly of split mFAP fragments. e Pre-assembled split mFAP competition model in which BCL2 is fused to m58 (BCL2_m58) (violet cartoon), aBFL1 is fused to m14 (m14_aBFL1) (orange cartoon), and fluorescence of DFHBI-1T (green spheres) is activated before unfused aBCL2 (yellow cartoon) competes with m14_aBFL1 for the BCL2-binding cleft of BCL2_m58 (arrow), resulting in fluorescence deactivation. f Temporal evolution of fluorescence fold-change in excess DFHBI-1T of pre-incubated equimolar BCL2_m58 and m14_aBFL1 at 2.00 µM final concentrations with unfused aBCL2 titrated in at (n = 1) 0 µM (black points), 4.00 µM (green points), and 10.0 µM (red points) final concentrations, showing the fits to a monophasic exponential function (lines) using non-linear least squares fitting. Source data is available for Fig. 6b, d, f.