Fig. 3: Observing binding modes of multiple proteins to a single substrate by multiwell plate smFRET.

a Competitive binding of RecA and SSB to ssDNA probed in a 96-well plate measurement by a combined variation of the concentration of SSB (0 to 50 nM) and RecA (0 to 2.6 µM) using the DNA construct dT₇₀ as a substrate. b E_FRET histograms of dT₇₀ in absence of proteins, presence of SSB at low and high concentration and RecA. The construct allows to identify unique E_FRET for unbound DNA, SSB₆₅ and SSB₃₅ binding modes, as well as a RecA filament on ssDNA. c Speciation curves as obtained from a multiwell plate smFRET experiment of dT₇₀ subjected to increasing concentration of SSB ranging from 0 to 4 µM. Data are presented as predicted value +/−68% CI as derived by the Gaussian fit. d 2-D E_FRET histogram of the competitive binding of RecA and SSB to dT₇₀ as obtained from a 96-well plate smFRET measurement. Bars and wedges on the left side depict RecA and SSB concentrations, respectively. Increasing RecA concentrations shift the transition to the SSB₆₅ binding to higher concentrations of SSB. e Fraction of molecules with FRET efficiency \(E\) > 0.4 versus [SSB] for increasing RecA concentrations. Fractions were fitted with an optimized 6-state equilibrium model (blue lines). Data are presented as mean +/− SD. The SD is derived from the counting uncertainty by simple error propagation. f 6-state model used to describe competitive binding of SSB and RecA to dT₇₀. T: empty dT₇₀; TS_m: SSB on dT₇₀ in SSB₆₅ binding mode; TS_m+n: SSB on dT₇₀ in SSB₃₅ binding mode; TR_p: RecA filament on dT₇₀; TR_qS_m: mixed state of SSB₆₅ and RecA on dT₇₀; and TR_rS_m+n: mixed state of SSB₃₅ and RecA on dT₇₀. The values \({K}_{{{{{{\rm{S}}}}}}}\), \({K}_{{{{{{\rm{S}}}}}}}^{*}\), \({K}_{{{{{{\rm{R}}}}}}}\), \({\alpha \cdot K}_{{{{{{\rm{R}}}}}}}\), \({\alpha \cdot \beta \cdot K}_{{{{{{\rm{R}}}}}}}\), \({\alpha \cdot K}_{{{{{{\rm{S}}}}}}}\), \(\beta \cdot {K}_{{{{{{\rm{S}}}}}}}^{*}\) denote the apparent dissociation constants with the corresponding Hill coefficients \(m\), \(n\), \(p\), \(q\), and \(r\). The RecA concentrations at half occupation (\({C}_{{{{{{\rm{i}}}}}},1/2}=\root i \of {K}\)) was extracted from the fit in panel E (blue lines). Values are given in the main text. Source data are provided as a Source data file.