Thermodynamic analysis of protein stability and ligand binding using a chemical modification- and mass spectrometry-based strategy.
Described here is a new technique, termed SPROX (stability of proteins from rates of oxidation), that can be used to measure the thermodynamic stability of proteins and protein-ligand complexes. SPROX utilizes hydrogen peroxide in the presence of increasing concentrations of a chemical denaturant to oxidize proteins. The extent of oxidation at a given oxidation time is determined as a function of the denaturant concentration using either electrospray or matrix-assisted laser desorption/ionization mass spectrometry. Ultimately, the denaturant concentration dependence of the oxidation reaction rate is used to evaluate a folding free energy (DeltaG(f)) and m value (deltaDeltaG(f)/delta[Den]) for the protein's folding/unfolding reaction. Measurements of such SPROX-derived DeltaG(f) and m values on proteins in the presence and absence of ligands can also be used to evaluate protein-ligand affinities (e.g., DeltaDeltaG(f) and Kd values). Presented here are SPROX results obtained on four model protein systems including ubiquitin, ribonuclease A (RNaseA), cyclophilin A (CypA), and bovine carbonic anhydrase II (BCAII). SPROX-derived DeltaG(f) and m values on these proteins are compared to values obtained using more established techniques (e.g., CD spectroscopy and SUPREX). The dissociation constants of several known protein-ligand complexes involving these proteins were also determined using SPROX and compared to previously reported values. The complexes included the CypA-cyclosporin A complex and the BCAII-4-carboxybenzenesulfonamide complex. The accuracy and precision of SPROX-derived thermodynamic parameters for the model proteins and protein-ligand complexes in this study are discussed as well as the caveats of the technique.
Duke Scholars
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Related Subject Headings
- Thermodynamics
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Sensitivity and Specificity
- Proteins
- Protein Folding
- Protein Denaturation
- Protein Binding
- Oxidation-Reduction
- Ligands
- Kinetics
Citation
Published In
DOI
EISSN
ISSN
Publication Date
Volume
Issue
Start / End Page
Related Subject Headings
- Thermodynamics
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
- Sensitivity and Specificity
- Proteins
- Protein Folding
- Protein Denaturation
- Protein Binding
- Oxidation-Reduction
- Ligands
- Kinetics