Conformational change in full-length mouse prion: a site-directed spin-labeling study.
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.
Inanami
Osamu
O
Laboratory 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
Hashida
Shukichi
S
Iizuka
Daisuke
D
Horiuchi
Motohiro
M
Hiraoka
Wakako
W
Shimoyama
Yuhei
Y
Nakamura
Hideo
H
Inagaki
Fuyuhiko
F
Kuwabara
Mikinori
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011328:Prions / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels
D011328:Prions; D011994:Recombinant Proteins; D013113:Spin Labels