Table 2:
Evaluation of KD and |Δωmax| values derived from 2D 1H,15N-HSQC titrations of 15N-PAR1GED when bound to GpIbα+PPACK-IIaa
| Peptide | Residue | KD for PPACK-IIa (μM) | |Δωmax| (ppm) | IIa residues in contact with PAR1 (49–62)9 |
|---|---|---|---|---|
| PAR1GED | E53 | KD too weak for NMR analysis | NA | In vicinity of T74, R75, and Y76 |
| PAR1GED+GpIbα | E53 | 126±36 | 0.32 ± 0.05 | In vicinity of T74, R75, and Y76 |
| PARIGED | D58 | 36±6.6 | 0.41 ± 0.05 | Not seen in X-ray (might contact R77a) |
| PAR1GED+GpIbα | D58 | 75±15 | 0.52 ± 0.05 | Not seen in X-ray (might contact R77a) |
a
For these 1H-15N-HSQC titrations, the PAR1 peptide concentrations were maintained at 50 μM and the PPACK-thrombin concentrations were diluted serially. KD values were determined from in-house scripts written in Python. Experimental data employed for these calculations included the various concentrations of protein and peptide. In addition, 15N NMR chemical shift differences for sets of free and bound conditions were utilized These HSQC titration series were performed at least in duplicate. A Monte-Carlo approach that assumes a 10% error in the serially diluted protein samples was employed for error analysis.