Advertisement

Simple Way to Detect Trp to Tb3+ Resonance Energy Transfer in Calcium-Binding Peptides Using Excitation Spectrum

  • Petra Lišková
  • Ivo Konopásek
  • Radovan FišerEmail author
SHORT COMMUNICATION
  • 53 Downloads

Abstract

The sensitized phosphorescence of Tb3+ is often used for the assessment of the ion binding to various chelating agents or natural Ca2+-binding proteins. The detailed structure of the Tb3+ excitation spectrum gives a special advantage for analysis; any extra absorption peak can be easily detected which provides simple and direct evidence that resonance energy transfer occurs. By employing the Tb3+ phosphorescence, we characterized the Ca2+-binding sites of two related peptides – self-processing module of the FrpC protein produced by bacterium Neisseria meningitidis and the shorter peptide derived from FrpC. Here we show that while the increase of direct Tb3+ excitation at 243 nm generally corresponds to Tb3+ association with various binding sites, the excitation enhancement in the 250–300 nm band signifies Tb3+-binding in the close proximity of aromatic residues. We demonstrate that the presence of resonance energy transfer could be easily detected by inspecting Tb3+ excitation spectra. Additionally, we show that the high level of specificity of Tb3+ steady state detection on the spectral level could be reached at very low Tb3+ concentrations by taking advantage of its narrow phosphorescence emission maximum at 545 nm and subtracting the averaged autofluorescence intensities outside this peak, namely at 525 and 565 nm.

Keywords

Terbium phosphorescence Tryptophan Protein fluorescence Energy transfer Excitation Spectrum Calcium-binding site 

Notes

Acknowledgements

This work was supported by project SVV 260426 of The Ministry of Education, Youth and Sports and the project 354611 (P.L.) of the Charles University. The authors wish to thank Lucie Jánská for excellent technical assistance and Dr. Zdeněk Fišar for borrowing the pulse diode.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Schlyer BD, Steel DG, Gafni A (Sep. 1995) Direct kinetic evidence for triplet state energy transfer from Escherichia coli alkaline phosphatase tryptophan 109 to bound terbium. J Biol Chem 270(39):22890–22894CrossRefGoogle Scholar
  2. 2.
    Basu G, Allen M, Willits D, Young M, Douglas T (Sep. 2003) Metal binding to cowpea chlorotic mottle virus using terbium(III) fluorescence. J Biol Inorg Chem 8(7):721–725CrossRefGoogle Scholar
  3. 3.
    Canada RG, Paltoo DN (Nov. 1998) Binding of terbium and cisplatin to C13* human ovarian cancer cells using time-resolved terbium luminescence. Biochim Biophys Acta-Mol Cell Res 1448(1):85–98CrossRefGoogle Scholar
  4. 4.
    Horrocks WD (1982) Lanthanide ion probes of biomolecular structure. Adv Inorg Biochem 4:201–261Google Scholar
  5. 5.
    Brittain HG, Richardson FS, Martin RB (1976) Terbium(III) emission as a probe of calcium(II) binding sites in proteins. J Am Chem Soc 98(25):8255–8260CrossRefGoogle Scholar
  6. 6.
    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer US, Boston, pp XXVI, 954.  https://doi.org/10.1007/978-0-387-46312-4 Google Scholar
  7. 7.
    Miller TL, Bennet LW, Spatz DS (1986) Terbium luminescence from complexes of angiotensin II, small peptides, and amino acids. Ohio J Sci 86(4):140–143Google Scholar
  8. 8.
    Jiao M et al (2013) Synthesis, structure and photoluminescence properties of europium-, terbium-, and thulium-doped Ca3Bi(PO4)3 phosphors. Dalton Trans 42(34):12395–12402CrossRefGoogle Scholar
  9. 9.
    Guo L, Yan B, Liu J-L, Sheng K, Wang X-L (2011) Coordination bonding construction, characterization and photoluminescence of ternary lanthanide (Eu3+, Tb3+) hybrids with phenylphenacyl-sulfoxide modified bridge and polymer units. Dalton Trans 40(3):632–638CrossRefGoogle Scholar
  10. 10.
    Tran TH, Tran KA, Hoang TK, Pham TH, Le QM (2012) Fabrication and properties of terbium phosphate nanorods. Adv Nat Sci Nanosci Nanotechnol 3(1):015010CrossRefGoogle Scholar
  11. 11.
    Ladokhin AS (2000) Fluorescence spectroscopy in peptide and protein analysis. In: Schoneich C (ed) Peptides and proteins (Encyclopedia of analytical chemistry: applications of instrumental methods). Wiley, New York.  https://doi.org/10.1002/9780470027318.a1611 CrossRefGoogle Scholar
  12. 12.
    Ohara P (1987) Lanthanide ions as luminescent probes of biomolecular structure. Photochem Photobiol 46(6):1067–1070CrossRefGoogle Scholar
  13. 13.
    DeJersey J, Morley P, Martin R (1981) Lanthanide probes in biological-systems - characterization of luminescence excitation-spectra of terbium complexes with proteins. Biophys Chem 13(3):233–243CrossRefGoogle Scholar
  14. 14.
    Dieke GH (Mar. 1970) Spectra and energy levels of rare earth ions in crystals. Am J Phys 38(3):399–400CrossRefGoogle Scholar
  15. 15.
    de Graaf D, Stelwagen SJ, Hintzen HT, de With G (Sep. 2003) Tb3+ luminescence as a tool to study clustering of lanthanide ions in oxynitride glasses. J Non-Cryst Solids 325(1):29–33CrossRefGoogle Scholar
  16. 16.
    Osicka R, Prochazkova K, Sulc M, Linhartova I, Havlicek V, Sebo P (Jun. 2004) A novel ‘clip-and-link’ activity of repeat in toxin (RTX) proteins from gram-negative pathogens - covalent protein cross-linking by an asp-Lys isopeptide bond upon calcium-dependent processing at an asp-pro bond. J Biol Chem 279(24):24944–24956CrossRefGoogle Scholar
  17. 17.
    Liskova PM et al (2016) Probing the Ca2+-assisted π-π interaction during Ca2+-dependent protein folding. Soft Matter 12(2):531–541CrossRefGoogle Scholar
  18. 18.
    Bala M et al (2018) Synthesis, photoluminescence behavior of green light emitting Tb(III) complexes and mechanistic investigation of energy transfer process. J Fluoresc 28(3):775–784CrossRefGoogle Scholar
  19. 19.
    Caldiño U, Speghini A, Bettinelli M (2006) Optical spectroscopy of zinc metaphosphate glasses activated by Ce3+ and Tb3+ ions. J Phys Condens Matter 18(13):3499–3508CrossRefGoogle Scholar
  20. 20.
    Neil ER, Fox MA, Pal R, Pålsson L-O, O’Sullivan BA, Parker D (2015) Chiral probe development for circularly polarised luminescence: comparative study of structural factors determining the degree of induced CPL with four heptacoordinate europium(III) complexes. Dalton Trans 44(33):14937–14951CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Genetics and Microbiology, Faculty of ScienceCharles UniversityPrague 2Czech Republic

Personalised recommendations