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Conformational change in full-length mouse prion: a site-directed spin-labeling study.

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Title: Conformational change in full-length mouse prion: a site-directed spin-labeling study.
Authors: Inanami, Osamu Browse this author →KAKEN DB
Hashida, Shukichi Browse this author
Iizuka, Daisuke Browse this author
Horiuchi, Motohiro Browse this author →KAKEN DB
Hiraoka, Wakako Browse this author
Shimoyama, Yuhei Browse this author
Nakamura, Hideo Browse this author →KAKEN DB
Inagaki, Fuyuhiko Browse this author →KAKEN DB
Kuwabara, Mikinori Browse this author
Keywords: Site-directed spin-labeling
Electron spin resonance
Conformational change
pH-sensitive region
Issue Date: 30-Sep-2005
Publisher: Elsevier Inc.
Journal Title: Biochemical and Biophysical Research Communications
Volume: 335
Issue: 3
Start Page: 785
End Page: 792
Publisher DOI: 10.1016/j.bbrc.2005.07.148
PMID: 16095563
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 Cu2+ 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 α-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 (τ) of 1.1, 3.3, and 4.8 ns were evaluated from the X-band ESR spectra at pH 7.4 and 20 °C for N96R1, D143R1, and T189R1, respectively. τ reflects the fact that the Cu2+ 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 °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 °C showed a gradual increase of τ from 2.9 to 4.8 ns. 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 Cu2+ binding region and D143 in Helix1 were conserved.
Type: article (author version)
Appears in Collections:獣医学院・獣医学研究院 (Graduate School of Veterinary Medicine / Faculty of Veterinary Medicine) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 稲波 修

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