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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- 作者：Petra L. Roulhac ; Kendall D. Powell ; Suraj Dhungana ; Katherine D. Weaver ; Timothy A. Mietzner ; Alvin L. Crumbliss ; and Michael C. Fitzgerald
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：December 21, 2004
- 年：2004
- 卷：43
- 期：50
- 页码：15767 - 15774
- 全文大小：127K
- 年卷期：v.43,no.50(December 21, 2004)
- ISSN：1520-4995
文摘
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 equilibriumunfolding/refolding properties of proteins and protein-ligand complexes. Here, we describe the applicationof SUPREX to the thermodynamic analysis of synergistic anion binding to iron-loaded ferric-bindingprotein (Fe3+FbpA-X, X = synergistic anion). The in vivo function of FbpA is to transport unchelatedFe3+ across the periplasmic space of certain Gram-negative bacteria, a process that requires simultaneousbinding of a synergistic anion. Our results indicate that Fe3+FbpA-X is not a so-called "ideal" proteinsystem for SUPREX analyses because it does not exhibit two-state folding properties and it does notexhibit EX2 H/D exchange behavior. However, despite these nonideal properties of the Fe3+FbpA-Xprotein-folding/unfolding reaction, we demonstrate that the SUPREX technique is still amenable to thequantitative thermodynamic analysis of synergistic anion binding to Fe3+FbpA. As part of this work, theSUPREX technique was used to evaluate the 
Gf values of four synergistic anion-containing complexesof Fe3+FbpA (i.e., Fe3+FbpA-PO4, Fe3+FbpA-citrate, Fe3+FbpA-AsO4, and Fe3+FbpA-SO4). The Gfvalue obtained for Fe3+FbpA-citrate relative to Fe3+FbpA-PO4 (1.45 ± 0.44 kcal/mol), is in goodagreement 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 measurementerror is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREXmethod for studying anion binding by FbpA, (ii) provides the first evaluation of a Gf value for Fe3+FbpA-SO4, -1.43 ± 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anionplays a role in controlling the lability of iron bound to FbpA in the transport process.
Gf values of four synergistic anion-containing complexesof Fe3+FbpA (i.e., Fe3+FbpA-PO4, Fe3+FbpA-citrate, Fe3+FbpA-AsO4, and Fe3+FbpA-SO4). The Gfvalue obtained for Fe3+FbpA-citrate relative to Fe3+FbpA-PO4 (1.45 ± 0.44 kcal/mol), is in goodagreement 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 measurementerror is very close to the magnitude of the value. This work (i) demonstrates the utility of the SUPREXmethod for studying anion binding by FbpA, (ii) provides the first evaluation of a Gf value for Fe3+FbpA-SO4, -1.43 ± 0.17 kcal/mol, and (iii) helps substantiate our hypothesis that the synergistic anionplays a role in controlling the lability of iron bound to FbpA in the transport process.