Skip to main content
SpringerLink
Log in

Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb

  • Original Paper
  • Published:
Journal of Fluorescence Aims and scope Submit manuscript

Abstract

The steady-state and time-resolved fluorescece spectroscopy is one of the most powerful method to detect and analyze subtle conformation change and interaction between peptide elements in protein. Phytocystatin Scb isolated from sunflower seeds includes a single Trp residue at position 85. In an attempt to investigate the interaction of the N-terminal region of Scb with the first and second hairpin loops by fluorescence spectroscopy of Trp residue, two Scb mutants in which single Trp locates at position 52 and 58, respectively, and their N-terminal removed mutants were generated. The N-terminal truncation changed the fluorescence decay kinetics of Trp52 from the triple exponential to double. Furthermore, the time-resolved fluorescence anisotropy residue indicated that the segmental motion of Trp52 was significantly enhanced by its N-terminal truncation. In contrast, Trp58 and Trp85 had little influence. The N-terminal successive truncations of Scb and its mutants resulted in the weaken inhibitors to papain. These results suggested that the N-terminal region of Scb interacts with the peptide segment preceding the first hairpin loop, thereby stabilizing the conformation of the hairpin loop structure.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

rScb:

recombinant sunflower cystatin b

Sca:

sunflower cystatin a

W85F/T52W:

recombinant sunflower cystatin b of which Trp85 and Thr52 are replaced with Phe and Trp, respectively

W85F/T58W:

recombinant sunflower cystatin b of which Trp85 and Thr58 are replaced with Phe and Trp, respectively

References

  1. Kondo H, Abe K, Nishimura I, Watanabe H, Emori Y, Arai S (1990) Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II. J Biol Chem 265:15832–15837

    PubMed  CAS  Google Scholar 

  2. Nagata K, Kudo N, Abe K, Arai S, Tanokura M (2000) Three-dimensional solution structure of oryzacystatin-I, a cysteine proteinase inhibitor of the rice, Oryza sativa L. japonica. Biochemistry 39:14753–14760

    Article  PubMed  CAS  Google Scholar 

  3. Barrett AJ, Rawlings ND, Davies ME, Machleidt W, Salvesen G, Turk V (1986) In Proteinase Inhibitors. Elsevier, Amsterdam, pp 515–569

    Google Scholar 

  4. Turk B, Turk V, Turk D (1997) Structural and functional aspects of papain-like cysteine proteinases and their protein inhibitors. Biol Chem 378:141–150

    PubMed  CAS  Google Scholar 

  5. Bode W, Enbh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V (1988) The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J 7:1939–1947

    Google Scholar 

  6. Stubbs MT, Laber B, Bode W, Huber R, Jerala R, Lenarcic B, Turk V (1990) The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J 9:1939–1947

    PubMed  CAS  Google Scholar 

  7. Pol E, Björk I (1999) Importance of the second binding loop and the C-terminal end of cystatin B (stefin B) for inhibition of cysteine proteinases. Biochemistry 38:10519–10526

    Article  PubMed  CAS  Google Scholar 

  8. Björk I, Brieditis I, Raub-Segall E, Pol E, Hakansson K, Abrahamson M (1996) The importance of the second hairpin loop of cystatin C for proteinase binding. Characterization of the interaction of Trp-106 variants of the inhibitor with cysteine proteinases. Biochemistry 35:10720–10726

    Article  PubMed  Google Scholar 

  9. Thiele U, Assfalg-machleidt I, Machleidt W, Auerswald EA (1990) N-terminal variants of recombinant stefin B: effect on affinity for papain and cathepsin B. Biol Chem Hopper-Seyler 371(Suppl.):125–136

    CAS  Google Scholar 

  10. Pol E, Björk I (2001) Role of the single cysteine residue, Cys 3, of human and bovine cystatin B (stefin B) in the inhibition of cysteine proteinases. Protein Sci 10:1729–1738

    Article  PubMed  CAS  Google Scholar 

  11. Abe K, Emori Y, Kondo H, Arai S, Suzuki K (1988) The NH2-terminal 21 amino acid residues are not essential for the papain-inhibitory activity of oryzacystatin, a member of the cystatin superfamily. Expression of oryzacystatin cDNA and its truncated fragments in Escherichia coli. J Biol Chem 263:7655–7659

    PubMed  CAS  Google Scholar 

  12. Urwin PE, Atkinson HJ, Mcpherson MJ (1995) Involvement of the NH2-terminal region of oryzacystatin-I in cysteine proteinase inhibition. Protein Eng 8:1303–1307

    Article  PubMed  CAS  Google Scholar 

  13. Estrada S, Pavlova A, Björk I (1999) The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L. Biochemistry 38:7339–7345

    Article  PubMed  CAS  Google Scholar 

  14. Shibuya K, Kaji H, Ohyama Y, Tate S, Kainosho M, Inagaki F, Samejima T (1995) Significance of the highly conserved Gly-4 residue in human cystatin A. J Biochem 118:635–642

    PubMed  CAS  Google Scholar 

  15. Hall A, Hakansson K, Mason RW, Grubb A, Abrahamson M (1995) Structural basis for the biological specificity of cystatin C. Identification of Leu-9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases. J Biol Chem 270:5115–5121

    Article  PubMed  CAS  Google Scholar 

  16. Mason RW, Sol-Church K, Abrahamson M (1998) Amino acid substitutions in the N-terminal segment of cystatin C create selective protein inhibitors of lysosomal cysteine proteinases. Biochem J 330:833–838

    PubMed  CAS  Google Scholar 

  17. Lindahl P, Ripoll D, Abrahamson M, Mort JS, Storer AC (1994) Evidence for the interaction of valine-10 in cystatin C with the S2 subsite of cathepsin B. Biochemistry 33:4384–4392

    Article  PubMed  CAS  Google Scholar 

  18. Estrada S, Olson ST, Raub-Segall E, Björk I (2000) The N-terminal region of cystatin A (stefin A) binds to papain subsequent to the two hairpin loops of the inhibitor. Demonstration of two-step binding by rapid-kinetic studies of cystatin A labeled at the N-terminus with a fluorescent reporter group. Protein Sci 9:2218–2224

    Article  PubMed  CAS  Google Scholar 

  19. Shubuya K, Kaji H, Itoh T, Ohyama Y, Tsujukami A, Tate S, Takeda A, Kumagai I, Hirao I, Miura K, Inagaki F, Samejima T (1995) Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity. Biochemistry 34:12185–12192

    Article  Google Scholar 

  20. Kouzuma Y, Kawano K, Kimura M, Yamasaki N, Kadowaki T, Yamamoto K (1996) Purification, characterization, and sequencing of two cysteine proteinase inhibitors, Sca and Scb, from sunflower (Helianthus annuus) seeds. J Biochem 119:1106–1113

    PubMed  CAS  Google Scholar 

  21. Doi-Kawano K, Kouzuma Y, Yamasaki N, Kimura M (1998) Molecular cloning, functional expression, and mutagenesis of cDNA encoding a cysteine proteinase inhibitor from sunflower seeds. J Biochem 124:911–916

    PubMed  CAS  Google Scholar 

  22. Deng WP, Nickoloff JA (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem 200:81–88

    Article  PubMed  CAS  Google Scholar 

  23. Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89

    Article  PubMed  CAS  Google Scholar 

  24. Yamashita S, Nishimoto E, Szabo AG, Yamasaki N (1996) Steady-state and time-resolved fluorescence studies on the ligand-induced conformational change in an active lysozyme derivative, Kyn62-lysozyme. Biochemistry 35:531–537

    Article  PubMed  CAS  Google Scholar 

  25. McKinnon AE, Szabo AG, Miller DR (1977) The deconvolution of photoluminescence data. J Phys Chem 81:1564–1570

    Article  CAS  Google Scholar 

  26. Gill PR, Murray W, Wright MH (1981) The Levenberg-Marquardt method. Practical optimization. Academic Press, London, pp 136–137

    Google Scholar 

  27. Willis KJ, Szabo AG (1992) Conformation of parathyroid hormone: time-resolved fluorescence studies. Biochemistry 31:8924–8931

    Article  PubMed  CAS  Google Scholar 

  28. Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2ndnd edn. Kluwer Academic/Plenum, New York

    Google Scholar 

  29. 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 

  30. 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

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, Hakozaki, Fukuoka, 812-8581, Japan

    Keiko Doi-Kawano, Yoshiaki Kouzuma & Makoto Kimura

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

    Etsuko Nishimoto, Daisuke Takahashi & Shoji Yamashita

Authors
  1. Keiko Doi-Kawano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Etsuko Nishimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yoshiaki Kouzuma
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Daisuke Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shoji Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Makoto Kimura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shoji Yamashita.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Doi-Kawano, K., Nishimoto, E., Kouzuma, Y. et al. Steady-State and Time-Resolved Fluorescence Spectroscopic Studies on Interaction of the N-terminal Region with the Hairpin Loop of the Phytocystain Scb. J Fluoresc 19, 631–639 (2009). https://doi.org/10.1007/s10895-008-0454-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10895-008-0454-7

Keywords

Search

Navigation