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.
(2013) Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry, mAbs, 5:1, 114-125,
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.
A protocol was developed to characterize the domain-specific thermodynamic stabilities of multidomain proteins using SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange). The protocol incorporates a protease digestion step into the conventional SUPREX protocol and enables folding free energy (DeltaGf) and cooperativity (m-value) measurements to be made on the individual domains of multidomain proteins in their native context (i.e., in the intact protein). Three multidomain protein systems (calmodulin, a Fyn construct, and transferrin) were used to validate the SUPREX-protease digestion protocol. The DeltaGf and m-value of each domain in the intact test proteins were measured in the absence and presence of ligands using the new protocol. Domain-specific thermodynamic parameters were obtained on each system; and the measured parameters were consistent with known biophysical properties of the test proteins. The known stabilization of the N-terminal domain of CaM in the context of the intact protein and the known binding affinity of a proline-rich peptide to the SH3 domain in the Fyn construct were successfully quantified using the new protocol. Qualitative information about the relative calcium binding affinities of the N- and C-terminal domains of CaM and about the relative iron binding affinities of the N- and C-terminal domains of transferrin was also obtained using the new protocol.
Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins in order to facilitate protein folding and prevent non-specific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports on the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H/D exchange), an H/D exchange and MALDI-based technique, to study the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding/unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX derived K d -values for Hsp33 complexes with four different substrates were found to be all within a range of 3-300 nM.Most proteins must fold into a well-defined three-dimensional structure in order to carry out their physiological roles within the cell. However, under different kinds of environmental stress conditions, such as elevated temperature, ultraviolet light, exposure of the cell to toxins (e.g., hypochlorous acid, arsenic, or trace metals) and infection, many proteins begin to lose their structure and function. Protein unfolding then often leads to non-specific aggregation, which is considered to be a largely irreversible process in vivo. Molecular chaperones are a class of proteins that function in the cell to recognize and selectively bind non-native proteins to prevent protein aggregation and facilitate their folding. It is generally believed that chaperones interact with unfolding protein substrates via hydrophobic interactions (for a review, see ref. (1)). For example, peptide binding studies using a number of different chaperones revealed that most show a greater preference toward hydrophobic peptides than to charged, hydrophilic peptides (2-4). Fluorescent hydrophobic probes have also been used to show that chaperones bind unfolded protein substrates upon exposure of hydrophobic surfaces (5-7). Finally, analysis of several X-ray crystallographic structures of chaperones indicated they preferentially bind to hydrophobic residues (4,8,9 Protein Expression and PurificationPurification of wild type Hsp33 and preparation of reduced inactive Hsp33 red was conducted as previously described (5). To activate Hsp33, 50 μM Hsp33 red was incubated with 500 μM NaOCl for 1 h at 30°C in 40 mM potassium phosphate buffer under constant shaking at 300 rpm. Excess NaOCl was removed using PD-10 desalt...
Table 2. Summary of guanidine-titration HDX-MS conditions of GCase. Conditions A [a] B [b] C [c] wt [mm] 3.0-R495H [mm] 3.0 3.0-N370S [mm] 3.0-3.0 No ligand +-IFG [mm]-214 (72 equiv) 214 (72 equiv) ABX [mm]-1150 (390 equiv) 1150 (390 equiv) GuHCl [m] 0.2-3.0 0.2-3.0 0.2-3.0 (0.5-5.4 with IFG) (0.5-5.4 with IFG) HEPES [mm] 50 50 50 pH read 7.0 7.0 7.0 DMSO [%] 0.3 0.3 0.3 D 2 O [%] 75 75 75 T [8C] 22 22 22 t inc [s] [d] 30 30 30 [a] Guanidine titration in the absence of ligand for wt, N370S, and R495H GCases. [b] Guanidine titration for R495H GCase in the IFG-or ABX-bound state. [c] Guanidine titration for N370S GCase in the IFG-or ABX-bound state. [d] Incubation time. 2162 www.chembiochem.org
B [b] C [c] wt [mm] 3.0 --R495H [mm] 3
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