Metal binding properties of single amino acid deletion mutants of zinc finger peptides: studies using cobalt(II) as a spectroscopic probe

Biophys J. 1993 Mar;64(3):749-53. doi: 10.1016/S0006-3495(93)81435-8.

Abstract

Peptides corresponding to Cys2His2 zinc finger domains from which one amino acid has been deleted have been synthesized and their metal-binding properties characterized. In contrast to earlier reports (Párraga, G., S. Horvath, L. Hood, E. T. Young, and R. E. Klevit. 1990. Proc. Natl. Acad. Sci. USA. 87:137-141.), such peptides do bind metal ions such as cobalt(II). A peptide with the sequence ProTyrLysCysProGluCysLysSerPheSerGlnLysSerAspLeuValLysHisGlnArgThrHis ThrGly (which corresponds to a previously characterized consensus zinc finger sequence from which a Gly residue immediately following the second Cys residue has been deleted) was found to form a 1:1 peptide to cobalt(II) complex with an absorption spectrum quite similar to those previously observed for zinc finger peptide-cobalt(II) complexes. The dissociation constant for this complex is 6 x 10(-6)M, a factor of 100 times higher than that for the parent peptide. A peptide with the sequence LysProTyrProCysGlyLeuCysArgCysPheThrArgArgAspLeuLeulleArgHisAlaGln - LyslleHisSerGlyAsnLeu corresponding to a similar mutation of the peptide ADR1 was also characterized. Spectroscopic studies with cobalt(II) revealed that this peptide forms both 1:1 and 2:1 peptide to cobalt(II) complexes. The absorption spectra of the two forms and the dissociation constants were determined via deconvolution methods. In contrast, the parent peptide ADR1a was found to form only a 1:1 complex under comparable conditions and this 1:1 complex was found to be more stable than that for the mutant. These results reveal that deletion mutations do adversely affect the stability of zinc finger peptide-metal complexes but that the effects are not as drastic as had been previously described.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Biophysical Phenomena
  • Biophysics
  • Cobalt
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Magnetic Resonance Spectroscopy
  • Metals / metabolism
  • Molecular Probes
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Saccharomyces cerevisiae Proteins*
  • Spectrophotometry
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Zinc Fingers / genetics
  • Zinc Fingers / physiology*

Substances

  • ADR1 protein, S cerevisiae
  • DNA-Binding Proteins
  • Metals
  • Molecular Probes
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Cobalt