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. Author manuscript; available in PMC: 2020 Feb 26.
Published in final edited form as: Biochemistry. 2019 Feb 11;58(8):1048–1060. doi: 10.1021/acs.biochem.8b00943

Table 3:

Evaluation of KD and |Δωmax| values derived from 2D 1H,15N-HSQC titrations for 15N labeled PAR3GEL when bound to GpIbα+PPACK-IIaa

Peptide Residue KD for PPACK-IIa (μM) |Δωmax| (ppm)28 IIa residues in contact with PAR3 (44–56)10
PAR3GEL28 L52 47 ± 6 1.84± 0.08 F34, L65, I82
PAR3GEL+GpIbα L52 KD too tight for NMR analysis NA F34, L65, I82
PAR3GEL28 E48 KD too tight for NMR analysis Insufficient data points R75
PAR3GEL+GpIbα E48 KD too tight for NMR analysis NA R75
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