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

作者：Kefeng Qin, Ying Yang, Peter Mastrangelo, David Westaway · 发表于：Journal of Biological Chemistry · 年份：2002 · DOI：10.1074/jbc.m108744200 · 被引用次数：141 · 研究领域：Prion Diseases and Protein Misfolding、Trace Elements in Health、Neurological diseases and metabolism

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.