Accuracy and precision of a new H/D exchange- and mass spectrometry-based technique for measuring the thermodynamic properties of protein-peptide complexes.
A new H/D exchange- and MALDI mass spectrometry-based technique, termed SUPREX, was used to characterize the thermodynamic properties of a series of model protein-peptide complexes of the Abelson tyrosine kinase SH3 domain (abl-SH3) and the S-Protein (S-Pro). The SUPREX technique was employed to evaluate the folding free energies (DeltaG(f) values) of each model protein in the absence and in the presence of a series of different peptide ligands. Ultimately, these SUPREX-derived DeltaG(f) values were used to calculate dissociation constants (K(d) values) for each of the nine protein-peptide complexes in this study. As part of this work, we describe a new data collection and analysis method that allows the accurate and precise determination of protein folding m-values in the SUPREX experiment. The m-values that we determined for the abl-SH3 domain and the S-Pro system were in good agreement with those determined by conventional techniques. Our results also indicate that the SUPREX-derived K(d) values for the protein-peptide complexes in this work were in reasonably good agreement with those determined by conventional techniques.
Powell
Kendall D
KD
Department of Chemistry, Duke University, Durham, North Carolina 27708-0346, USA.
Fitzgerald
Michael C
MC
D000595:Amino Acid Sequence; D002151:Calorimetry; D003903:Deuterium; D017666:Deuterium Oxide; D006859:Hydrogen; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics; D014508:Urea; D018909:src Homology Domains
D008024:Ligands; D010446:Peptide Fragments; D006859:Hydrogen; D014508:Urea; D003903:Deuterium; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C022542:ribonuclease S; D017666:Deuterium Oxide