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投稿者: Yuma Morimoto

Article

Robust Affinity Standards for Cu(I) Biochemistry

† School of Chemistry and Biochemistry, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, 901 Atlantic Drive, Atlanta, Georgia 30332, United States
‡ X-ray Crystallography Center, Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, Georgia 30322, United States
J. Am. Chem. Soc., 2013, 135 (49), pp 18549–18559
DOI: 10.1021/ja408827d
Publication Date (Web): December 3, 2013
Copyright © 2013 American Chemical Society

Abstract

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The measurement of reliable Cu(I) protein binding affinities requires competing reference ligands with similar binding strengths; however, the literature on such reference ligands is not only sparse but often conflicting. To address this deficiency, we have created and characterized a series of water-soluble monovalent copper ligands, MCL-1, MCL-2, and MCL-3, that form well-defined, air-stable, and colorless complexes with Cu(I) in aqueous solution. X-ray structural data, electrochemical measurements, and an extensive network of equilibrium titrations showed that all three ligands form discrete Cu(I) complexes with 1:1 stoichiometry and are capable of buffering Cu(I) concentrations between 10–10 and 10–17 M. As most Cu(I) protein affinities have been obtained from competition experiments with bathocuproine disulfonate or 2,2′-bicinchoninic acid, we further calibrated their Cu(I) stability constants against the MCL series. To demonstrate the application of these reagents, we determined the Cu(I) binding affinity of CusF (log K = 14.3 ± 0.1), a periplasmic metalloprotein required for the detoxification of elevated copper levels in Escherichia coli. Altogether, this interconnected set of affinity standards establishes a reliable foundation that will facilitate the precise determination of Cu(I) binding affinities of proteins and small-molecule ligands.
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