Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards

Xiao, Zhiguang, Brose, Jens, Schimo, Sonja, Ackland, Susan M., La Fontaine, Sharon and Wedd, Anthony G. 2011, Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards, Journal of biological chemistry, vol. 286, no. 13, pp. 11047-11055.

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Title Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins : Detection probes and affinity standards
Author(s) Xiao, Zhiguang
Brose, Jens
Schimo, Sonja
Ackland, Susan M.
La Fontaine, Sharon
Wedd, Anthony G.
Journal name Journal of biological chemistry
Volume number 286
Issue number 13
Start page 11047
End page 11055
Total pages 9
Publisher American Society for Biochemistry and Molecular Biology
Place of publication Bethesda, Md.
Publication date 2011-04-01
ISSN 0021-9258
1083-351X
Summary Literature estimates of metal-protein affinities are widely scattered for many systems, as highlighted by the class of metallo-chaperone proteins, which includes human Atox1. The discrepancies may be attributed to unreliable detection probes and/or inconsistent affinity standards. In this study, application of the four CuI ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their CuI affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with CuI to yield distinct 1:2 chromatophoric complexes [CuIL2] 3- with formation constants β2 = 1017.2 and 1019.8 M-2, respectively. These constants do not depend on proton concentration for pH ≥7.0. Consequently, they are a pair of complementary and stable probes capable of detecting free Cu+ concentrations from 10-12 to 10-19 M. Dtt binds CuI with KD∼10-15 M at pH 7, but it is air-sensitive, and its CuI affinity varies with pH. The CuI binding properties of Atox1 and related proteins (including the fifth and sixth domains at the N terminus of the Wilson protein ATP7B) were assessed with these probes. The results demonstrate the following: (i) their use permits the stoichiometry of high affinity CuI binding and the individual quantitative affinities (KD values) to be determined reliably via noncompetitive and competitive reactions, respectively; (ii) the scattered literature values are unified by using reliable probes on a unified scale; and (iii) Atox1-type proteins bind CuI with sub-femtomolar affinities, consistent with tight control of labile Cu+ concentrations in living cells.
Language eng
Field of Research 030499 Medicinal and Biomolecular Chemistry not elsewhere classified
Socio Economic Objective 970103 Expanding Knowledge in the Chemical Sciences
HERDC Research category C1 Refereed article in a scholarly journal
Copyright notice ©2011, American Society for Biochemistry and Molecular Biology
Persistent URL http://hdl.handle.net/10536/DRO/DU:30044168

Document type: Journal Article
Collection: School of Life and Environmental Sciences
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