Thermodynamic stability measurements on multimeric proteins using a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based method.

Journal Article

We recently reported on a new H/D exchange- and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V(a)L(d) trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopy-based methods.

Full Text

Duke Authors

Cited Authors

  • Powell, KD; Wales, TE; Fitzgerald, MC

Published Date

  • April 2002

Published In

Volume / Issue

  • 11 / 4

Start / End Page

  • 841 - 851

PubMed ID

  • 11910027

International Standard Serial Number (ISSN)

  • 0961-8368

Digital Object Identifier (DOI)

  • 10.1110/ps.3820102

Language

  • eng

Conference Location

  • United States