Figure 4.

Binding affinities and modelling of complex formation. (A) ELISAs showing the ability of FLAG-tagged Ldb1_LID to bind to the cut Isl1/Ldb1_LID and Lhx3/Ldb1_LID complexes. Data were corrected for background binding to GST, and the error bars show the range of values obtained from triplicates of a single experiment. The inset shows a schematic of tethered intramolecular constructs of the Isl1–Ldb1_LID, where the linker contains a Factor Xa protease site. After treatment with Factor Xa, the linker is cut to form an intermolecular complex. (B) GdnHCl denaturation curves for Lhx3–Ldb1_LID and Lhx3–Isl1_LBD constructs as indicated. (C–E) Trends for the formation of protein and protein:DNA complexes containing Ldb1, Lhx3 and Isl1. Concentrations of all components were set at 1, 10 and 100 nM. (C) Distribution of Ldb1/LIM-HD complexes using the binary model (no interaction between Isl1 and Lhx3, and no DNA). (D) Distribution of protein-only complexes using the ternary model, which also considers binding of Isl1 and Lhx3. (E) Distribution of key protein:DNA complexes relative to Ldb1/Lhx3/DNA. The I series shows the results of using the independent model, the SA series shows the results of the single-molecule DNA-binding advantage model where the binding of two and four HDs to a single molecule of DNA shows increased binding affinities over single HD binding.