Copper refolding of prion protein.
We have shown previously that normal mouse prion protein (MoPrP) binds copper ions during protein refolding and acquires antioxidant activity. In this report, we probe the structure of the copper refolded form of MoPrP to determine how copper binding alters the secondary and tertiary features of the protein. Circular dichroism showed that recombinant MoPrP prepared in the presence of copper (as Cu(++)) showed an increased signal in the 210-220 nm range of the spectrum. Changes in protein conformation were localised to the N-terminal region of MoPrP using a panel of antibodies to assess epitope accessibility. The copper refolded recombinant prion protein had reduced proteinase K (PK) sensitivity when compared to the non-copper liganded form. Reduced PK sensitivity was not due to aggregation however as high resolution electron microscopy showed a homogenous preparation with little aggregate when compared to the non-copper form. Finally, disruption of the single disulphide linkage in MoPrP significantly diminished the antioxidant activity of the copper refolded form suggesting that activity was not solely dependent on bound copper but also on a conformation enabled by the formation of the disulphide bond.
Wong
B S
BS
School of Animal and Microbial Sciences, Reading, RG6 6AH, United Kingdom.
Vénien-Bryan
C
C
Williamson
R A
RA
Burton
D R
DR
Gambetti
P
P
Sy
M S
MS
Brown
D R
DR
Jones
I M
IM
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003300:Copper / Q000378:metabolism* / Q000494:pharmacology*; D004220:Disulfides / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D008854:Microscopy, Electron; D011328:Prions / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis; D013482:Superoxide Dismutase / Q000378:metabolism
D000906:Antibodies; D000975:Antioxidants; D004220:Disulfides; D011328:Prions; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003300:Copper; D013482:Superoxide Dismutase; D019286:Endopeptidase K