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

Journal of Fluorescence volume 23, pages 407–415 (2013)Cite this article

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

References

  1. Royer CA (2006) Probing protein folding and conformational transition with fluorescence. Chem Rev 106:1769–1784

    PubMed  Article  CAS  Google Scholar 

  2. Santra MK, Banerjee A, Krishnakumar SS, Rahaman O, Panda D (2004) Multiple-probe analysis of folding and unfolding pathways of human serum albumin: evidence for a framework mechanism of folding. Eur J Biochem 271:1789–1797

    PubMed  Article  CAS  Google Scholar 

  3. Ferguson N, Day R, Johnson CM, Allen MD, Daggett V, Fersht AR (2005) Simulation and experiment at high temperatures: ultrafast folding of a thermophilic protein by nucleation-condensation. J Mol Biol 347:855–870

    PubMed  Article  CAS  Google Scholar 

  4. Vendruscolo M, Paci E, Dobson CM, Karplus M (2001) Three key residues form a critical contact network in a protein folding transition state. Nature 409:641–645

    PubMed  Article  CAS  Google Scholar 

  5. Au TK, Collins RA, Lam TL, Ng TB, Fong WP, Wan DCC (2000) The plant ribosome inactivating proteins luffin and saporin are potent inhibitors of HIV-1 integrase. FEBS Lett 471:169–172

    PubMed  Article  CAS  Google Scholar 

  6. Wang Y-X, Jacob J, Wingfield PT, Palmer I, Stahl SJ, Kaufman JD, Huang PL, Huang PL, Lee-Huang S, Torchia DA (2000) Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and β-sheet topology with the A chain of ricin, a type-II RIP. Protein Sci 9:138–144

    PubMed  Article  CAS  Google Scholar 

  7. Corrado G, Bovi PD, Ciliento R, Gaudio L, Maro AD, Aceto S, Lorito M, Rao R (2005) Inducible expression of a Phytolacca heterotepala ribosome-inactivating protein leads to enhanced resistance against major fungal pathogens in tobacco. Phytopathology 95:206–215

    PubMed  Article  CAS  Google Scholar 

  8. Park S-W, Stevens NM, Vivanco JM (2002) Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes, and correlation with antifungal activity. Planta 216:227–234

    PubMed  Article  CAS  Google Scholar 

  9. Endo Y, Mitsui K, Mochizuki M, Tsurugi K (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem 262:5908–5912

    PubMed  CAS  Google Scholar 

  10. Fukunaga Y, Nishimoto E, Otosu T, Murakami Y, Yamashita S (2008) The unfolding of α-momorcharin proceeds through the compact folded intermediate. J Biochem 144:457–466

    PubMed  Article  CAS  Google Scholar 

  11. Fukunaga Y, Nishimoto E, Yamashita K, Otosu T, Yamashita S (2007) The partially unfolded state of β-momorcharin characterized with steady-state and time-resolved fluorescence studies. J Biochem 141:9–18

    PubMed  Article  CAS  Google Scholar 

  12. Honjo E, Watanabe K (1999) Expression of mature pokeweed antiviral protein with or without C-terminal extrapeptide in Escherichia coli as a fusion with maltose-binding protein. Biosci Biotechnol Biochem 63:1291–1294

    PubMed  Article  CAS  Google Scholar 

  13. Iizasa E, Nagano Y (2006) Highly efficient yeast-based in vivo DNA cloning of multiple DNA fragments and the simultaneous construction of yeast/Escherichia coli shuttle vectors. Biotechniques 40:79–83

    PubMed  Article  CAS  Google Scholar 

  14. Marykwas DL, Passmore SE (1995) Mapping by multifragment cloning in vivo. Proc Natl Acad Sci USA 92:11701–11705

    PubMed  Article  CAS  Google Scholar 

  15. Willis KJ, Szabo AG (1989) Resolution of tyrosyl and tryptophyl fluorescence emission from subtilisins. Biochemistry 28:4902–4908

    Article  CAS  Google Scholar 

  16. Zuker M, Szabo AG, Bramall L, Krajcarski DT, Selinger B (1985) Delta function convolution method (DFCM) for fluorescence decay experiments. Rev Sci Instrum 56:14–22

    Article  CAS  Google Scholar 

  17. Otosu T, Nishimoto E, Yamashita S (2009) Fluorescence decay characteristics of indole compounds revealed by time-resolved area-normalized emission spectroscopy. J Phys Chem A 113:2847–2853

    PubMed  Article  CAS  Google Scholar 

  18. Lakowicz JR, Laczko G, Gryczynski I (1987) Picosecond resolution of tyrosine fluorescence and anisotropy decays by 2-GHz frequency-domain fluorometry. Biochemistry 26:82–90

    PubMed  Article  CAS  Google Scholar 

  19. Eisinger J, Feuer B, Lamola AA (1969) Intramolecular singlet excitation transfer. Applications to polypeptides. Biochemistry 8:3908–3915

    PubMed  Article  CAS  Google Scholar 

  20. Oishi O, Yamashita S, Nishimoto E, Lee S, Sugihara G, Ohno M (1997) Conformations and orientations of aromatic amino acid residues of tachyplesin I in phospholipid membranes. Biochemistry 36:4352–4359

    PubMed  Article  CAS  Google Scholar 

  21. Monzingo AF, Collins EJ, Ernst SR, Irvin JD, Robertus JD (1993) The 2.5 Å structure of pokeweed antiviral protein. J Mol Biol 233:705–715

    PubMed  Article  CAS  Google Scholar 

  22. Lakowicz JR (2006) Advanced anisotropy concepts. In: Principles of fluorescence spectroscopy, 3rd edn. Springer, New York, pp 413–441

  23. Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 2. Analysis of experimental results. J Am Chem Soc 104:4559–4570

    Article  CAS  Google Scholar 

  24. Nishimoto E, Yamashita S, Szabo AG, Imoto T (1998) Internal motion of lysozyme studied by time-resolved fluorescence depolarization of tryptophan residues. Biochemistry 37:5599–5607

    PubMed  Article  CAS  Google Scholar 

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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

Keywords

  • Protein folding/unfolding
  • FRET
  • Fluorescence depolarization
  • Ribosome-inactivating protein
  • Pokeweed anti-viral protein