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

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• 作者：Binbin Gong ; Adriana Ramos ; Ester Va虂zquez-Ferna虂ndez ; Christopher J. Silva ; Jana Alonso ; Zengshan Liu ; Jesu虂s R. Requena
• 刊名：Biochemistry
• 出版年：2011
• 出版时间：June 7, 2011
• 年：2011
• 卷：50
• 期：22
• 页码：4963-4972
• 全文大小：872K
• 年卷期：v.50,no.22(June 7, 2011)
• ISSN：1520-4995

文摘

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鈥?31) [rSHaPrP(90鈥?31)] and SHaPrP 27鈥?0, 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鈥揜₂₂₉ (containing Y₂₂₅ and Y₂₂₆), which in rSHaPrP(90鈥?31) was much more extensively modified than in SHaPrP 27鈥?0; peptide H₁₁₁鈥揜₁₃₆, containing Y₁₂₈, was also more modified in rSHaPrP(90鈥?31). Conversely, peptides Y₁₄₉鈥揜₁₅₁, Y₁₅₇鈥揜₁₆₄, and R₁₅₁鈥揧₁₆₂ suffered more extensive modification in SHaPrP 27鈥?0. Acetic anhydride modified very extensively peptide G₉₀鈥揔₁₀₆, containing K₁₀₁, K₁₀₄, K₁₀₆, and the amino terminus, in both rSHaPrP(90鈥?31) and SHaPrP 27鈥?0. These results suggest that (1) SHaPrP 27鈥?0 exhibits important conformational differences in the C-terminal region with respect to rSHaPrP(90鈥?31), 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鈥?0. (2) On the other hand, tyrosines contained in the stretch spanning approximately Y₁₄₉鈥揜₁₆₄ are more accessible in SHaPrP 27鈥?0, suggesting rearrangements in 伪-helix H1 and the short 尾-sheet of rSHaPrP(90鈭?31). (3) The amino-terminal region of SHaPrP 27鈥?0 is very accessible. These data should help in the validation and construction of structural models of PrP^Sc.