Unification of the copper(I) binding affinities of the metallo-chaperones Atx1, Atox1, and related proteins: detection probes and affinity standards.
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 Cu(I) ligand probes bicinchoninate, bathocuproine disulfonate, dithiothreitol (Dtt), and glutathione (GSH) is reviewed, and their Cu(I) affinities are re-estimated and unified. Excess bicinchoninate or bathocuproine disulfonate reacts with Cu(I) to yield distinct 1:2 chromatophoric complexes [Cu(I)L(2)](3-) with formation constants β(2) = 10(17.2) and 10(19.8) 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 Cu(I) with K(D) ∼10(-15) m at pH 7, but it is air-sensitive, and its Cu(I) affinity varies with pH. The Cu(I) 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 Cu(I) binding and the individual quantitative affinities (K(D) 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 Cu(I) with sub-femtomolar affinities, consistent with tight control of labile Cu(+) concentrations in living cells.
School of Chemistry and Bio21 Institute, University of Melbourne, Victoria, Australia. z.xiao@unimelb.edu.au
Zhiguang
Z
Xiao
Jens
J
Brose
Sonja
S
Schimo
Susan M
SM
Ackland
Sharon
S
La Fontaine
Anthony G
AG
Wedd
D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D000079922:Copper Transport Proteins; D006801:Humans; D056511:Metallochaperones; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011804:Quinolines / Q000737:chemistry*
C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D011804:Quinolines; D003300:Copper
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