Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique
- Laboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita 18-Jo Nishi 9-chome, Sapporo 060-0818 (Japan)
- Laboratory of Prion Diseases, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818 (Japan)
- Laboratory of Biophysics, School of Science and Technology, Meiji University, Kawasaki 214-8571 (Japan)
- Soft-Matter Physics Laboratory, Graduate School of Emergent Science, Muroran Institute of Technology, Muroran 050-8585 (Japan)
- Laboratory of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0818 (Japan)
We analyzed the pH-induced mobility changes in moPrP{sup C} {alpha}-helix and {beta}-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of {alpha}-helix1 (H1, codon 143-151), four amino acid residues of {beta}-sheet1 (S1, codon 127-130), and four amino acid residues of {beta}-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrP{sup C} (moPrP{sup C}) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/{delta}H of the central ({sup 14}N hyperfine) component (M{sub I} = 0) in the ESR spectrum of spin-labeled moPrP{sup C} 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 PrP{sup C} to PrP{sup Sc} in acidic organelles such as the endosome.
- OSTI ID:
- 20854579
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 350, Issue 3; Other Information: DOI: 10.1016/j.bbrc.2006.09.082; PII: S0006-291X(06)02110-3; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
Similar Records
Instability of familial spongiform encephalopathy-related prion mutants
Proximity of thiol esters and bait region in human. cap alpha. /sub 2/-macroglobulin: paramagnetic mapping