SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange) Analysis of the Thermodynamics of Synergistic Anion Binding by Ferric-Binding Protein (FbpA), a Bacterial Transferrin

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Abstract
SUPREX (stability of unpurified proteins from rates of H/D exchange) is a H/D exchange- and matrix-assisted laser desorption/ionization (MALDI)-based technique for characterizing the equilibrium unfolding/refolding properties of proteins and protein−ligand complexes. Here, we describe the application of SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-binding protein (Fe3+FbpA−X, X = synergistic anion). The in vivo function of FbpA is to transport unchelated Fe3+ across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneous binding of a synergistic anion. Our results indicate that Fe3+FbpA−X is not a so-called “ideal” protein system for SUPREX analyses because it does not exhibit two-state folding properties and it does not exhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe3+FbpA−X protein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to the quantitative thermodynamic analysis of synergistic anion binding to Fe3+FbpA. As part of this work, the SUPREX technique was used to evaluate the ΔΔGf values of four synergistic anion-containing complexes of Fe3+FbpA (i.e., Fe3+FbpA−PO4, Fe3+FbpA−citrate, Fe3+FbpA−AsO4, and Fe3+FbpA−SO4). The ΔΔGf value obtained for Fe3+FbpA−citrate relative to Fe3+FbpA−PO4 (1.45 ± 0.44 kcal/mol), is in good agreement with that reported previously (1.98 kcal/mol). The value obtained for Fe3+FbpA−AsO4 (0.58 ± 0.45 kcal/mol) was also consistent with that reported previously (0.68 kcal/mol), but the measurement error is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREX method for studying anion binding by FbpA, (ii) provides the first evaluation of a ΔΔGf value for Fe3+FbpA−SO4, −1.43 ± 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anion plays a role in controlling the lability of iron bound to FbpA in the transport process.