Because previous experiments have shown that hPPARα binds LCFA and LCFA-CoA with high affinity and at a single binding site, equal molarities of hPPARα and ligand were mixed and allowed to bind prior to elucidation of hPPARα's affinity for hLXRα.
The Δ G/Δ G m value for these binding sites was equal to 90.9% and 90.7%, respectively.
At the root of the tree, we assumed that the number of binding sites was equal to the equilibrium-binding site number.
Crucially, when a mix of all three mutants is substituted into the system, such that the molarity of PCNA1, PCNA2 and PCNA3 binding sites is equal to WT but all three sites are never on the same PCNA molecule, processing activity is not restored to WT levels, rather the maximal activity we observe is the same as the activity of the least impaired mutant PCNA ring (ΔP3).
Thus, the number of positive examples from an annotated binding site equals the number of amino acids in the binding site.
In fact, an unpublished ChIP-seq analysis (Bilke S, Kauer M, Kovar H, Meltzer P, personal communication), which increases the number of sequencing reads for EWS/FLI1 more than 30-fold over the previously published Guillon et al. study [ 47], identified almost equal amounts of EWS/FLI1 binding sites close to transcriptional start sites of genes and in intragenic regions (over 4 kb from the TSS).
A bis-ANS solution (10 μmol, equal to twice the amount required to saturate the binding sites) was added to a 0.1 mg/mL peptide solution in phosphate buffer (pH 7.2).
To analyze the binding data, we used equation (1), describing a 1 : 1 interaction between one TRAF2 monomer and one GSTP1-1 subunithethe binding sites being equal and independent (see Materials and Methods section).
The equilibrium dissociation constants (Kd) of the complexes were calculated by assuming a 1 : 1 interaction between one TRAF2 monomer and one GSTP1-1 subunithethe binding sites being equal and independent.
Since the vast majority of known binding sites correspond to only one TF each in a given organism, one may assume that the TF family size is approximately equal to the number of DNA binding domains used by that family.
