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.
West
Graham M
GM
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Tang
Liangjie
L
Fitzgerald
Michael C
MC
D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D007700:Kinetics; D008024:Ligands; D010084:Oxidation-Reduction; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013816:Thermodynamics
D008024:Ligands; D011506:Proteins