Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique.
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We analyzed the pH-induced mobility changes in moPrPC α-helix and β-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of α-helix1 (H1, codon 143–151), four amino acid residues of β-sheet1 (S1, codon 127–130), and four amino acid residues of β-sheet2 (S2, codon 160–163) were substituted for by cysteine residues. These recombinant mouse PrPC (moPrPC) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/δH of the central (14N hyperfine) component (MI = 0) in the ESR spectrum of spin-labeled moPrPC was measured as a mobility parameter of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the H1 region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the S1 region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e., (1) the N-terminal tertiary contact site of H1, (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of H1 was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrPC to PrPSc in acidic organelles such as the endosome.
- Elsevier Inc.の論文
- 2006-11-24
Elsevier Inc. | 論文
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