title: Robust affinity standards for Cu(I) biochemistry.
abstract: 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.
authors.affiliation: School of Chemistry and Biochemistry, Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology , 901 Atlantic Drive, Atlanta, Georgia 30332, United States.
authors.forename: Pritha
authors.initials: P
authors.lastname: Bagchi
authors.forename: M Thomas
authors.initials: MT
authors.lastname: Morgan
authors.forename: John
authors.initials: J
authors.lastname: Bacsa
authors.forename: Christoph J
authors.initials: CJ
authors.lastname: Fahrni
mesh_terms: D003300:Copper / Q000737:chemistry*; D008024:Ligands; D008958:Models, Molecular; D014961:X-Ray Diffraction
chemical_list: D008024:Ligands; D003300:Copper