Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry

Randy M. Whittal, Haydn L. Ball, Fred E. Cohen, Alma L. Burlingame, Stanley B. Prusiner, Michael A. Baldwin

Research output: Contribution to journalArticle

221 Citations (Scopus)

Abstract

Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N- terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.

Original languageEnglish (US)
Pages (from-to)332-343
Number of pages12
JournalProtein Science
Volume9
Issue number2
StatePublished - 2000

Fingerprint

Mass spectrometry
Copper
Mass Spectrometry
Peptides
Ions
Electrospray ionization
Electrospray Ionization Mass Spectrometry
Stoichiometry
Acetylation
Metals
Prions
Prion Proteins
Complexation
Histidine
Metal ions
Endosomes
Binding Sites
Circular Dichroism
Lysosomes

Keywords

  • Circular dichroism
  • Copper binding
  • Dissociation constants
  • Electrospray ionization mass spectrometry
  • Prion protein
  • PrP
  • PrP peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Whittal, R. M., Ball, H. L., Cohen, F. E., Burlingame, A. L., Prusiner, S. B., & Baldwin, M. A. (2000). Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Science, 9(2), 332-343.

Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. / Whittal, Randy M.; Ball, Haydn L.; Cohen, Fred E.; Burlingame, Alma L.; Prusiner, Stanley B.; Baldwin, Michael A.

In: Protein Science, Vol. 9, No. 2, 2000, p. 332-343.

Research output: Contribution to journalArticle

Whittal, RM, Ball, HL, Cohen, FE, Burlingame, AL, Prusiner, SB & Baldwin, MA 2000, 'Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry', Protein Science, vol. 9, no. 2, pp. 332-343.
Whittal RM, Ball HL, Cohen FE, Burlingame AL, Prusiner SB, Baldwin MA. Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. Protein Science. 2000;9(2):332-343.
Whittal, Randy M. ; Ball, Haydn L. ; Cohen, Fred E. ; Burlingame, Alma L. ; Prusiner, Stanley B. ; Baldwin, Michael A. / Copper binding to octarepeat peptides of the prion protein monitored by mass spectrometry. In: Protein Science. 2000 ; Vol. 9, No. 2. pp. 332-343.
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AU - Prusiner, Stanley B.

AU - Baldwin, Michael A.

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N2 - Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N- terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.

AB - Electrospray ionization mass spectrometry (ESI-MS) was used to measure the binding of Cu2+ ions to synthetic peptides corresponding to sections of the sequence of the mature prion protein (PrP). ESI-MS demonstrates that Cu2+ is unique among divalent metal ions in binding to PrP and defines the location of the major Cu2+ binding site as the octarepeat region in the N- terminal domain, containing multiple copies of the repeat ProHisGlyGlyGlyTrpGlyGln. The stoichiometries of the complexes measured directly by ESI-MS are pH dependent: a peptide containing four octarepeats chelates two Cu2+ ions at pH 6 but four at pH 7.4. At the higher pH, the binding of multiple Cu2+ ions occurs with a high degree of cooperativity for peptides C-terminally extended to incorporate a fifth histidine. Dissociation constants for each Cu2+ ion binding to the octarepeat peptides, reported here for the first time, are mostly in the low micromolar range; for the addition of the third and fourth Cu2+ ions to the extended peptides at pH 7.4, K(D)'s are <100 nM. N-terminal acetylation of the peptides caused some reduction in the stoichiometry of binding at both pH's. Cu2+ also binds to a peptide corresponding to the extreme N-terminus of PrP that precedes the octarepeats, arguing that this region of the sequence may also make a contribution to the Cu2+ complexation. Although the structure of the four-octarepeat peptide is not affected by pH changes in the absence of Cu2+, as judged by circular dichroism, Cu2+ binding induces a modest change at pH 6 and a major structural perturbation at pH 7.4. It is possible that PrP functions as a Cu2+ transporter by binding Cu2+ ions from the extracellular medium under physiologic conditions and then releasing some or all of this metal upon exposure to acidic pH in endosomes or secondary lysosomes.

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