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Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence

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Abstract

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state.

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Abbreviations

PAP:

Pokeweed anti-viral protein

GuHCl:

Guanidine hydrochloride

RIP:

Ribosome-inactivating protein

FRET:

Fluorescence resonance energy transfer

TCSPC:

Time-correlated single photon counting

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Authors and Affiliations

  1. Industrial Technology Center of Nagasaki, 2-1303-8, Ikeda, Ohmura, Nagasaki, 856-0026, Japan

    Shuzo Matsumoto

  2. Institute of Biophysics, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Fukuoka, 812-8581, Japan

    Yuka Taniguchi, Hiromichi Nakashima, Shoji Yamashita & Etsuko Nishimoto

  3. Institute for Material Chemistry and Engineering, Kyushu University, Kasuga, Fukuoka, 816-8580, Japan

    Yukihiro Fukunaga

  4. Department of Applied Biological Sciences, Saga University, Honjo, Saga, 840-8502, Japan

    Keiichi Watanabe

Authors
  1. Shuzo Matsumoto
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  2. Yuka Taniguchi
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  3. Yukihiro Fukunaga
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  4. Hiromichi Nakashima
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  5. Keiichi Watanabe
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  6. Shoji Yamashita
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  7. Etsuko Nishimoto
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Correspondence to Etsuko Nishimoto.

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Matsumoto, S., Taniguchi, Y., Fukunaga, Y. et al. Structural Characteristic of Folding/Unfolding Intermediate of Pokeweed Anti-viral Protein Revealed by Time-resolved Fluorescence. J Fluoresc 23, 407–415 (2013). https://doi.org/10.1007/s10895-013-1155-4

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  • DOI: https://doi.org/10.1007/s10895-013-1155-4

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