H/D Exchange- and Mass Spectrometry-Based Strategy for the Thermodynamic Analysis of Protein--Ligand Binding

摘要

The equilibrium unfolding properties of four model proteinsystems were characterized using SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is an H/D exchange- and mass spectrometry-based technique for measuring the free energy ((DELTA)G_(f)) and m-value ((delta)(DELTA)G_(f)/(delta)[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-x_(L)) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (>approx15 kDa) or multi-domain proteins. In the absence of ligand, (DELTA)G_(f) and m-values for these proteins could not be evaluated using the conventional data acquisition and analysis methods previously established for SUPREX. However, ligand-bound forms of the proteins were amenable to conventional SUPREX analyses, and it was possible to evaluate reasonably accurate and precise binding free energies of selected ligands. In some cases, protein-ligand dissociation constants (K_(d) values) could also be ascertained. The SUPREX-derived binding free energies and K_(d) values evaluated here were in good agreement with those reported on the same complexes using other techniques.