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Published on: January 8, 2015
Conformational change in full-length mouse prion: a site-directed spin-labeling study
Osamu Inanami1, Shukichi Hashida, Daisuke Iizuka
1Laboratory of Radiation Biology, Department of Environmental Veterinary Medical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. inanami@vetmed.hokudai.ac.jp
Abstract:
The structure of the mouse prion (moPrP) was studied using site-directed spin-labeling electron spin resonance (SDSL-ESR). Since a previous NMR study by Hornemanna et al., [Hornemanna, Korthb, Oeschb, Rieka, Widera, Wüthricha, Glockshubera, Recombinant full-length murine prion protein, mPrP (23-231): purification and spectroscopic characterization, FEBS Lett. 413 (1997) 277-281] has indicated that N96, D143, and T189 in moPrP are localized in a Cu(2+) binding region, Helix1 and Helix2, respectively, three recombinant moPrP mutations (N96C, D143C, and T189C) were expressed in an Escherichia coli system, and then refolded by dialysis under low pH and purified by reverse-phase HPLC. By using the preparation, we succeeded in preserving a target cystein residue without alteration of the alpha-helix structure of moPrP and were able to apply SDSL-ESR with a methane thiosulfonate spin label to the full-length prion protein. The rotational correlation times (tau) of 1.1, 3.3, and 4.8ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 degrees C for N96R1, D143R1, and T189R1, respectively. tau reflects the fact that the Cu(2+) binding region is more flexible than Helix1 or Helix2. ESR spectra recorded at various temperatures revealed two phases together with a transition point at around 20 degrees C in D143R1 and T189R1, but not in N96R1. With the variation of pH from 4.0 to 7.8, ESR spectra of T189R1 at 20 degrees C showed a gradual increase of tau from 2.9 to 4.8ns. On the other hand, the pH-dependent conformational changes in N96R1 and D143R1 were negligible. These results indicated that T189 located in Helix2 possessed a structure sensitive to physiological pH changes; simultaneously, N96 in the Cu(2+) binding region and D143 in Helix1 were conserved.
Insights
Site-directed spin-labeling electron spin resonance (SDSL-ESR) revealed distinct structural dynamics in mouse prion protein (moPrP). The T189 residue in Helix2 showed pH-sensitive structural changes, unlike N96 and D143.
Area of Science:
- Structural biology
- Biophysics
- Prion protein research
Background:
- Previous Nuclear Magnetic Resonance (NMR) studies identified a Cu(2+) binding region and specific residues (N96, D143, T189) in mouse prion protein (moPrP).
- Understanding the structural dynamics of moPrP is crucial for elucidating prion disease mechanisms.
Purpose of the Study:
- To investigate the structural dynamics and flexibility of specific regions within full-length mouse prion protein (moPrP) using site-directed spin-labeling electron spin resonance (SDSL-ESR).
- To determine the influence of pH and temperature on the structural integrity of moPrP mutants.
Main Methods:
- Expression and refolding of three recombinant moPrP mutations (N96C, D143C, T189C) in Escherichia coli.
- Purification using reverse-phase High-Performance Liquid Chromatography (HPLC).
- Application of SDSL-ESR with a methane thiosulfonate spin label to analyze rotational correlation times (tau) and conformational changes.
Main Results:
- Rotational correlation times (tau) indicated that the Cu(2+) binding region (N96) is more flexible than Helix1 (D143) and Helix2 (T189).
- Electron spin resonance (ESR) spectra revealed temperature-dependent phase transitions around 20°C for D143R1 and T189R1, but not N96R1.
- T189R1 exhibited significant pH-dependent structural changes, with tau increasing from 2.9 to 4.8 ns as pH varied from 4.0 to 7.8, while N96R1 and D143R1 showed negligible changes.
Conclusions:
- The residue T189, located in Helix2 of moPrP, possesses a structure sensitive to physiological pH variations.
- Residues N96 (Cu(2+) binding region) and D143 (Helix1) exhibit conserved structural characteristics across the studied pH range.
- SDSL-ESR is effective in characterizing the structural dynamics of full-length prion proteins and identifying pH-sensitive regions.
