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Biochemistry. 2011 Jun 7;50(22):4963-72. doi: 10.1021/bi102073j. Epub 2011 May 11.

Probing structural differences between PrP(C) and PrP(Sc) by surface nitration and acetylation: evidence of conformational change in the C-terminus.

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Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, PR China.


We used two chemical modifiers, tetranitromethane (TNM) and acetic anhydride (Ac(2)O), 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 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 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).

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