Probing structural differences between PrPC and PrPSc by surface nitration and acetylation: evidence of conformational change in the C-terminus

We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride (Ac2O), which specifically target accessible tyrosine and lysine residues, respectively, to modify recombinant Syrian hamster PrP(90-231) [rSHaPrP(90-231)] and SHaPrP 27-30, the proteinase K-resistant core of PrPSc isolated from brain of scrapie-infected Syrian hamsters. Our aim was to find locations of conformational change. Modified proteins were subjected to in-gel proteolytic digestion with trypsin or chymotrypsin and subsequent analysis by mass spectrometry (MALDI-TOF). Several differences in chemical reactivity were observed. With TNM, the most conspicuous reactivity difference seen involves peptide E-221-R-229 (containing Y-225 and Y-226), which in rSHaPrP(90-231) was much more extensively modified than in SHaPrP 27-30; peptide H-111-R-136, containing Y-128, was also more modified in rSHaPrP(90-231). Conversely, peptides Y-149-R-151, Y-157-R-164, and R-151-Y-162 suffered more extensive modification in SHaPrP 27-30. Acetic anhydride modified very extensively peptide G(90)-K-106, containing K-101, K-104, K-106, and the amino terminus, in both rSHaPrP(90-231) and SHaPrP 27-30. These results suggest that (1) SHaPrP 27-30 exhibits important conformational differences in the C-terminal region with respect to rSHaPrP(90-231), resulting in the loss of solvent accessibility of Y-225 and Y-226, very solvent-exposed in the latter conformation; because other results suggest preservation of the two C-terminal helices, this might mean that these are tightly packed in SHaPrP 27-30. (2) On the other hand, tyrosines contained in the stretch spanning approximately Y-149-R-164 are more accessible in SHaPrP 27-30, suggesting rearrangements in alpha-helix HI and the short beta-sheet of rSHaPrP(90-231). (3) The amino-terminal region of SHaPrP 27-30 is very accessible. These data should help in the validation and construction of structural models of PrPSc.