Figure 6:

Determination of Binding Affinity (K_D) for ¹⁵N-labeled F47 and D54 of PAR3G_FD interacting with Prothrombin and PPACK-IIa. For this NMR titration series, the peptide ligand concentration was kept constant and the ProT and PPACK-IIa concentrations were serially diluted. As a result, the NMR titrations were measuring the binding of protein to a defined peptide concentration. (A) Interactions between ProT and PAR3G ¹⁵N-F47 led to a K_D = 64 ± 8 μM, (B) ProT and PAR3G ¹⁵N-D54 led to a K_D = 65 ± 12 μM, and (C) PPACK-IIa and PAR3G ¹⁵N-F47 led to a K_D = 40 ± 10 μM. NMR titrations were done in triplicate. The reported K_D values were determined using in-house scripts written using Python. The term |Δδ_obs| ¹⁵Nppm = δ^15N_Bound - δ^15Ν _Free reflects the absolute difference in chemical shift between the bound and free states of the particular ¹⁵N-residue. Error analysis was carried out using a Monte-Carlo approach assuming a 10% error in the serially diluted protein samples. See Materials and Methods for more details.