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

Abstract

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).

The structure of the infectious prion protein

“…Mass spectrometry revealed that tyrosines Y225 and Y226 become less exposed in Syrian hamster PrP 27-30, while being completely accessible to small reagents in PrP C . 39 This supports the notion that a major rearrangement of the C-terminus takes places during conversion of PrP C to PrP Sc . Also, tyrosines Y162 and/or Y163 become exposed in PrP Sc .…”

Selective Amplification of Classical and Atypical Prions Using Modified Protein Misfolding Cyclic Amplification

“…Indeed, the gradual shift in mobility of SSLOW LMW products during dgPMCAb supports the adaptation hypothesis. It is noteworthy that previous studies using mass spectrometry identified several internal PK cleavage sites including residues 139, 142, and 154 in 263K and Drowsy brain-derived PrP Sc upon treatment with high concentrations of PK and/or exposure to partially denaturing conditions (39,40). Adaptation of PrP Sc to dgPMCAb might involve changes in its quaternary structure leading to an increase in accessibility of internal sites.…”

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

“…9 These results meant that the same amino acid could react differently with the same reagent, depending on whether it was in the PrP C Sc has been reacted with commercially available reagents to obtain structural information about its C-terminal region. 96 Hamster PrP Sc was reacted with tetranitromethane (TNM) or acetic anhydride (Ac 2 O), which nitrated the tyrosine or acetylated the ε-amino groups of lysine, respectively. After reaction, the samples were analyzed by mass spectrometry to determine which tyrosine or lysine was covalently modified.…”

“…In this way antibodies were used to detect covalent modifications of PrP Sc . 96 The lysines present in the PrP C conformer reacted completely with synthetic reagents. The extent of this reaction was monitored by Western blot using antibodies that did and did not have lysinecontaining epitopes.…”