Table 1:

Evaluation of K_D and |Δω_max| values derived from 2D ¹H,¹⁵N-HSQC titrations of ¹⁵N-labeled PAR1G_FD, PAR1G_KE, and PAR1G_ED bound to PPACK-Thrombin^a

Peptide	Residue	KD for PPACK-IIa (μM)	|Δωmax| (ppm)	IIa residues in contact with PAR1 (49–62)
PAR1GFD	F55	251	NA	F34, L65, 182
PAR1GFD	D50	Solvent exchange issue	NA	R73
PAR1GKE	K51	167 ± 49	0.09 ± 0.02	In vicinity of R73
PAR1GKE	E60	280	NA	Not seen in X-ray (might contact I82, K110)
PAR1GED	E53	Too weak for KD calculation	NA	T74, Y76, and R75
PAR1GED	D58	36 ± 6.6	0.41 ± 0.05	Not seen in X-ray (might contact R77a)

^aFor these HSQC titrations, the PAR1 peptide concentrations were maintained at 50 μM while the PPACK-thrombin concentrations were diluted serially. K_D 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 chemical shift differences for sets of free and bound conditions were utilized for F55, E60, and D58. ¹H chemical shift differences were employed for the K51. The HSQC titrations were carried out at least in duplicate. A Monte-Carlo approach that assumes a 10% error in the serially diluted protein samples was employed for error analysis.