Journal of Fluorescence

, 19:631

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

  • Keiko Doi-Kawano
  • Etsuko Nishimoto
  • Yoshiaki Kouzuma
  • Daisuke Takahashi
  • Shoji Yamashita
  • Makoto Kimura
Original Paper

DOI: 10.1007/s10895-008-0454-7

Cite this article as:
Doi-Kawano, K., Nishimoto, E., Kouzuma, Y. et al. J Fluoresc (2009) 19: 631. doi:10.1007/s10895-008-0454-7

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.

Keywords

Proteinase inhibitorCystatinTime-resolved fluorescenceFluorescence depolarizationSingle Trp protein

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

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Keiko Doi-Kawano
    • 1
  • Etsuko Nishimoto
    • 2
  • Yoshiaki Kouzuma
    • 1
  • Daisuke Takahashi
    • 2
  • Shoji Yamashita
    • 2
  • Makoto Kimura
    • 1
  1. 1.Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of AgricultureGraduate School of Kyushu UniversityFukuokaJapan
  2. 2.Institute of Biophysics, Department of Bioscience and Biotechnology, Faculty of AgricultureGraduate School of Kyushu UniversityFukuokaJapan