Figure 7:

1D and 2D ¹H-¹⁵N HSQC NMR titrations of PAR3G_FD (44–56) in the presence of PPACK-R77aA. All NMR samples were in 25mM H₃PO₄, 150 mM NaCl, 0.2 mM EDTA and 10 % D₂O (pH 6.5). (A) For the 1D HSQC NMR titrations, the starting complexes included 50 μΜ PAR3G_FD (44– 56, ¹⁵N-F47, ¹⁵N-D54) in 150 μM PPACK-R77aA. The serial dilutions resulted in PPACK-R77aA to PAR3G_FD ratios tliat spanned from 3:1 to 0.1:1. Note that at the start of titration (3:1 protein to peptide) the amide proton for PAR3G F47 was at 8.3 ppm and for PAR3G D54 at 8.2 ppm. By a ratio of (0.7 to 1), the two peaks overlapped into a single peak. As the PPACK-R77aA thrombin was further diluted, the F47 and D54 peaks contained to change resonance positions and eventually matched those of the free PAR3G_FD peptide. Representative data sets are shown. (B) For the 2D ¹H-¹⁵N HSQC titrations, starting complexes included 50 μM PAR3G_FD (44–56,¹⁵N-F47,¹⁵N-D54) in 150 μΜ PPACK-R77aA. The serial dilutions resulted in PPACK-thrombin to PAR3G_FD ratios that spanned from 3:1 to 0.1:1. Representative data sets are shown. Colors for the 2D ‘Η-^Ν HSQC crosspeaks span from blue (highest protein-peptide ratio) to red (free peptide).