JBIC Journal of Biological Inorganic Chemistry

, Volume 18, Issue 6, pp 669–678 | Cite as

Spectroscopic characterization of copper(I) binding to apo and metal-reconstituted zinc finger peptides

  • Reginald T. Doku
  • Grace Park
  • Korin E. Wheeler
  • Kathryn E. Splan
Original Paper

Abstract

Cu(I) exhibits high affinity for thiolate ligands, suggesting that thiol-rich zinc or iron binding sites may be subject to disruption during copper stress conditions. Zinc fingers constitute a large class of metalloproteins that use a combination of cysteine and histidine residues that bind Zn(II) as a structural element. Despite the shared preference of both copper and zinc for thiolate and amine coordination, the susceptibility of zinc finger domains toward copper substitution is not well studied. We report spectroscopic studies that characterize the Cu(I) binding properties of the zinc finger consensus peptides CP-CCHH, CP-CCHC, and CP-CCCC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Cu(I) binds to both the apopeptides and the Co(II)-substituted peptides, and the stoichiometry of Cu(I) binding is dependent on the number of cysteine thiols at the metal binding site. Fluorescence studies of the Zn(II)–NCp7_C complex indicate that Cu(I) also effectively competes with Zn(II) at the metal binding site, despite the high affinity of Zn(II) for the CCHC binding motif. Circular dichroism studies on both CP-CCHC and NCp7_C show that the conformations of the Cu(I)-bound complexes differ substantially from those of the Zn(II) species, implying that Cu(I) substitution is likely to impact zinc finger function. These results show that for the peptides studied here, Cu(I) is the thermodynamically favored metal despite the known high Zn(II) affinity of zinc finger domains, suggesting that Cu(I)-substituted zinc finger domains might be relevant in the context of both copper toxicity mechanisms and copper-responsive transcription factors.

Keywords

Zinc fingers Copper binding peptides Electronic absorption spectroscopy Metal ion toxicity 

Abbreviations

BCA

Bicinchoninate

CD

Circular dichroism

DTNB

5,5′-Dithiobis(2-nitrobenzoic acid)

NCp7

Nucleocapsid protein p7 of human immunodeficiency virus type 1

NCp7_C

C-terminal zinc finger domain of NCp7

ZF

Zinc finger

Supplementary material

775_2013_1012_MOESM1_ESM.pdf (204 kb)
Supplementary Material Electronic absorption data for Cu(I) and Co(II) complexes, titration spectra of Co(II)-substituted peptides with Zn(II), and binding constant data for FluoZin-1 is available as electronic supplementary material. (PDF 204 kb)

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Copyright information

© SBIC 2013

Authors and Affiliations

  • Reginald T. Doku
    • 1
  • Grace Park
    • 2
  • Korin E. Wheeler
    • 2
  • Kathryn E. Splan
    • 1
  1. 1.Department of ChemistryMacalester CollegeSaint PaulUSA
  2. 2.Department of Chemistry and BiochemistrySanta Clara UniversitySanta ClaraUSA

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