Copper-catalyzed oxidation of the recombinant SHa(29-231) prion protein

Metal-catalyzed oxidation may result in structural damage to proteins and h as been implicated in aging and disease, including neurological disorders s uch as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidize d protein molecules. PrP contains a histidine-rich octarepeat domain that b inds copper. Because copper-binding histidine residues are particularly pro ne to metal-catalyzed oxidation, we investigated the effect of this reactio n on the recombinant prion protein SHaPrP(29-231), Using Cu2+/ascorbate, we oxidized SHaPrP(29-231) in vitro. Oxidation was demonstrated by liquid chr omatography/mass spectrometry, which showed the appearance of protein speci es of higher mass, including increases in multiples of 16, characteristic o f oxygen incorporation. Digestion studies using Lys C indicate that the 29- 101 region, which includes the histidine-containing octarepeats, is particu larly affected by oxidation. Oxidation was time- and copper concentration-d ependent and was evident with copper concentrations as low as 1 muM. Concom itant with oxidation, SHaPrP(29-231) suffered aggregation and precipitation , which was nearly complete after 15 min, when the prion protein was incuba ted at 37 degreesC with a 6-fold molar excess of Cu2+. These findings indic ate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structur al transition leading to aggregation.