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Updated: Jul 19, 2026
Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique
Yasuko Watanabe1, Osamu Inanami, Motohiro Horiuchi
1Laboratory 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.
Abstract:
We analyzed the pH-induced mobility changes in moPrP(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(C) (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/deltaH of the central (14N hyperfine) component (M(I) = 0) in the ESR spectrum of spin-labeled moPrP(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(C) to PrP(Sc) in acidic organelles such as the endosome.
Insights
This study used spin labeling to map pH-induced structural changes in mouse prion protein (moPrP(C)). The N-terminal region of helix 1 (H1) is most sensitive to pH, potentially explaining PrP(C) to PrP(Sc) conversion in acidic organelles.
Area of Science:
- Structural Biology
- Biophysics
- Prion Biology
Background:
- The cellular prion protein (PrP(C)) undergoes conformational changes to form infectious PrP(Sc) isoforms, a process implicated in neurodegenerative diseases.
- Understanding the structural dynamics of PrP(C) in response to environmental factors like pH is crucial for elucidating the conversion mechanism.
- Previous studies suggested involvement of specific regions, but direct evidence of pH-induced mobility changes was lacking.
Purpose of the Study:
- To investigate pH-induced changes in the mobility of alpha-helix and beta-sheet regions within mouse PrP(C) (moPrP(C)).
- To identify specific pH-sensitive sites within moPrP(C) that may be involved in the conversion process.
- To correlate structural mobility with the three-dimensional structure of moPrP(C) under varying pH conditions.
Main Methods:
- Cysteine-scanning site-directed spin labeling (SDSL) was employed to introduce spin labels at specific residues within alpha-helix 1 (H1), beta-sheet 1 (S1), and beta-sheet 2 (S2) of moPrP(C).
- Electron spin resonance (ESR) spectroscopy was used to measure the mobility of the attached nitroxide spin labels (R1) as a function of pH.
- Mobility parameters were analyzed in relation to known structural features and previously identified tertiary contact sites.
Main Results:
- Mobility analysis revealed distinct patterns correlating with the helical and buried nature of H1, S1, and S2 regions.
- Three key pH-sensitive sites were identified: the N-terminal tertiary contact site of H1, the C-terminal end of H1, and the S2 region.
- The N-terminal tertiary contact region of H1 exhibited the highest pH sensitivity, readily becoming flexible upon a slight decrease in pH.
Conclusions:
- The mobility of spin-labeled residues in moPrP(C) accurately reflects its three-dimensional structure.
- The N-terminal tertiary contact site of H1 is a critical, highly pH-sensitive region within moPrP(C).
- These findings provide molecular evidence supporting the role of acidic organelles, like endosomes, in initiating the conversion of PrP(C) to PrP(Sc).
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