title: 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).
authors.affiliation: Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, PR China.
authors.forename: Binbin
authors.initials: B
authors.lastname: Gong
authors.forename: Adriana
authors.initials: A
authors.lastname: Ramos
authors.forename: Ester
authors.initials: E
authors.lastname: Vázquez-Fernández
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Silva
authors.forename: Jana
authors.initials: J
authors.lastname: Alonso
authors.forename: Zengshan
authors.initials: Z
authors.lastname: Liu
authors.forename: Jesús R
authors.initials: JR
authors.lastname: Requena
mesh_terms: D000107:Acetylation; D000818:Animals; D006224:Cricetinae; D008647:Mesocricetus; D018621:PrPC Proteins / Q000737:chemistry* / Q000378:metabolism; D018620:PrPSc Proteins / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D018621:PrPC Proteins; D018620:PrPSc Proteins; D014357:Trypsin