Probing structural differences between PrP^C and PrP^Sc 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 PrP ^Sc 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 E₂₂₁-R₂₂₉ (containing Y ₂₂₅ and Y₂₂₆), which in rSHaPrP(90-231) was much more extensively modified than in SHaPrP 27-30; peptide H₁₁₁-R ₁₃₆, containing Y₁₂₈, was also more modified in rSHaPrP(90-231). Conversely, peptides Y₁₄₉-R₁₅₁, Y ₁₅₇-R₁₆₄, and R₁₅₁-Y₁₆₂ suffered more extensive modification in SHaPrP 27-30. Acetic anhydride modified very extensively peptide G₉₀-K₁₀₆, containing K₁₀₁, K₁₀₄, K₁₀₆, 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 Y₂₂₅ and Y₂₂₆, 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-R₁₆₄ are more accessible in SHaPrP 27-30, suggesting rearrangements in α-helix H1 and the short β-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 PrP^Sc.