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J. Biol. Chem., Vol. 283, Issue 4, 1870-1881, January 25, 2008

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Deconvoluting the Cu²⁺ Binding Modes of Full-length Prion Protein^*

Mark Klewpatinond¹, Paul Davies, Suzanne Bowen, David R. Brown, and John H. Viles²

From the School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London, E1 4NS and the Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom

The prion protein (PrP) is a cell-surface Cu²⁺-binding glycoprotein that when misfolded is responsible for a number of transmissible spongiform encephalopathies. Full-length PrP-(23–231) and constructs in which the octarepeat region has been removed, or His⁹⁵ and His¹¹⁰ is replaced by alanine residues, have been used to elucidate the order and mode of Cu²⁺ coordination to PrP-(23–231). We have built on our understanding of the appearance of visible CD spectra and EPR for various PrP fragments to characterize Cu²⁺ coordination to full-length PrP. At physiological pH, Cu²⁺ initially binds to full-length PrP in the amyloidogenic region between the octarepeats and the structured domain at His⁹⁵ and His¹¹⁰. Only subsequent Cu²⁺ ions bind to single histidine residues within the octarepeat region. Ni²⁺ ions are used to further probe metal binding and, like Cu²⁺, Ni²⁺ will bind individually to His⁹⁵ and His¹¹⁰, involving preceding main chain amides. Competitive chelators are used to determine the affinity of the first mole equivalent of Cu²⁺ bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu²⁺ binding sites support the suggestion that PrP could act as a sacrificial quencher of free radicals generated by copper redox cycling.

Received for publication, October 11, 2007 , and in revised form, November 26, 2007.

^* This work was supported in part by Biotechnology and Biological Sciences Research Council Project grants. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Supported by a Biotechnology and Biological Sciences Research Council Studentship.

² To whom correspondence should be addressed. Fax: 44-0-20-8983-8443; E-mail: j.viles{at}qmul.ac.uk.

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