H/D exchange- and mass spectrometry-based strategy for the thermodynamic analysis of protein-ligand binding.

Journal Article

The equilibrium unfolding properties of four model protein systems 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 (DeltaGf) and m-value (deltaDeltaGf/delta[denaturant]) associated with the folding/unfolding reaction of a protein. The model proteins in this study (calmodulin, carbonic anhydrase II, RmlB, Bcl-xL) were chosen to test the applicability of SUPREX to the thermodynamic analysis of larger (> approximately 15 kDa) or multidomain proteins. In the absence of ligand, DeltaGf 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 (Kd values) could also be ascertained. The SUPREX-derived binding free energies and Kd values evaluated here were in good agreement with those reported on the same complexes using other techniques.

Full Text

Duke Authors

Cited Authors

  • Tang, L; Hopper, ED; Tong, Y; Sadowsky, JD; Peterson, KJ; Gellman, SH; Fitzgerald, MC

Published Date

  • August 1, 2007

Published In

Volume / Issue

  • 79 / 15

Start / End Page

  • 5869 - 5877

PubMed ID

  • 17580981

International Standard Serial Number (ISSN)

  • 0003-2700

Digital Object Identifier (DOI)

  • 10.1021/ac0700777

Language

  • eng

Conference Location

  • United States