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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
Biochemical and Biophysical Research Communications
|August 13, 2005
Summary
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.