A set of robust fluorescent peptide probes for quantification of Cu(II) binding affinities in the micromolar to femtomolar range

作者：Young Tessa R; Wijekoon Chathuri J K; Spyrou Benjamin; Donnelly Paul S; Wedd Anthony G; Xiao Zhiguang^*

来源：Metallomics, 2015, 7(3): 567-578.

DOI：10.1039/c4mt00301b

Reliable quantification of copper binding affinities and identification of the binding sites provide a molecular basis for an understanding of the nutritional roles and toxic effects of copper ions. Sets of chromophoric probes are now available that can quantify Cu(I) binding affinities from nanomolar to attomolar concentrations on a unified scale under in vitro conditions. Equivalent probes for Cu(II) are lacking. This work reports development of a set of four fluorescent dansyl peptide probes (DP1-4) that can quantify Cu(II) binding affinities from micromolar to femtomolar concentrations, also on a unified scale. The probes were constructed by conjugation of a dansyl group to four short peptides of specific design. Each was characterised by its dissociation constant K-D, its pH dependence and the nature of its binding site. One equivalent of Cu(II) is bound by the individual probes that display different and well-separated affinities at pH 7.4 (logK(D) = -8.1, -10.1, -12.3 and -14.1, respectively). Intense fluorescence is emitted at lambda(max) similar to 550 nm upon excitation at similar to 330 nm. Binding of Cu(II) quenches the fluorescence intensity linearly until one equivalent of Cu(II) is bound. Multiple approaches and multiple affinity standards were employed to ensure reliability. Selected examples of application to well-characterised Cu(II) binding peptides and proteins are presented. These include A beta 16 peptides, two naturally occurring Cu(II)-chelating motifs in human serum and cerebrospinal fluid with sequences GHK and DAHK and two copper binding proteins, CopC from Pseudomonas syringae and PcoC from Escherichia coli. Previously reported affinities are reproduced, demonstrating that peptides DP1-4 form a set of robust and reliable probes for Cu(II) binding to peptides and protein targets.

• 出版日期2015