title: Mapping Cu(II) binding sites in prion proteins by diethyl pyrocarbonate modification and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometric footprinting.
abstract: Although Cu(II) ions bind to the prion protein (PrP), there have been conflicting findings concerning the number and location of binding sites. We have combined diethyl pyrocarbonate (DEPC)-mediated carbethoxylation, protease digestion, and mass spectrometric analysis of apo-PrP and copper-coordinated mouse PrP23-231 to "footprint" histidine-dependent Cu(II) coordination sites within this molecule. At pH 7.4 Cu(II) protected five histidine residues from DEPC modification. No protection was afforded by Ca(II), Mn(II), or Mg(II) ions, and only one or two residues were protected by Zn(II) or Ni(II) ions. Post-source decay mapping of DEPC-modified histidines pinpointed residues 60, 68, 76, and 84 within the four PHGGG/SWGQ octarepeat units and residue 95 within the related sequence GGGTHNQ. Besides defining a copper site within the protease-resistant core of PrP, our findings suggest application of DEPC footprinting methodologies to probe copper occupancy and pathogenesis-associated conformational changes in PrP purified from tissue samples.
authors.affiliation: Centre for Research in Neurodegenerative Diseases, the Department of Laboratory Medicine and Pathobiology, and the Mass Spectrometry Laboratory, Molecular Medicine Research Centre, University of Toronto, Toronto, Ontario M5S 3H2, Canada.
authors.forename: Kefeng
authors.initials: K
authors.lastname: Qin
authors.forename: Ying
authors.initials: Y
authors.lastname: Yang
authors.forename: Peter
authors.initials: P
authors.lastname: Mastrangelo
authors.forename: David
authors.initials: D
authors.lastname: Westaway
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D003300:Copper / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism
chemical_list: D011328:Prions; D003300:Copper; D014357:Trypsin; D004047:Diethyl Pyrocarbonate