Skip to main content
SpringerLink
Log in

Probe-dependence of competitive fluorescent ligand binding assays to odorant-binding proteins

  • Communication
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers.

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

References

  1. Pert CB, Pasternak G, Snyder SH. Opiate agonists and antagonists discriminated by receptor binding in brain. Science. 1973;182:1359–61.

    CAS  PubMed  Google Scholar 

  2. Snyder SH. Receptors, neurotransmitters and drug responses. N Engl J Med. 1979;300:465–72.

    CAS  PubMed  Google Scholar 

  3. Snyder SH. Opiate receptors and beyond: 30 years of neural signaling research. Neuropharmacology. 2004;47(Suppl. 1):274–85.

    CAS  PubMed  Google Scholar 

  4. Bruns RF, Lawson-Wendling K, Pugsley TA. A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Anal Biochem. 1983;132:74–81.

    CAS  PubMed  Google Scholar 

  5. Paolini S, Tanfani F, Fini C, Bertoli E, Pelosi P. Porcine odorant-binding protein: structural stability and ligand affinities measured by Fourier-transform infrared spectroscopy and fluorescence spectroscopy. Biochim Biophys Acta. 1999;1431:179–88.

    CAS  PubMed  Google Scholar 

  6. Pelosi P, Zhu J, Knoll W. From radioactive ligands to biosensors: binding methods with olfactory proteins. Appl Microbiol Biotechnol. 2018;102:8213–27.

    CAS  PubMed  Google Scholar 

  7. Fisher HF, Singh N. Calorimetric methods for interpreting protein-ligand interactions. Methods Enzymol. 1995;259:194–221.

    CAS  PubMed  Google Scholar 

  8. Burova TV, Choiset Y, Jankowski CK, Haertlé T. Conformational stability and binding properties of porcine odorant binding protein. Biochemistry. 1999;38:15043–51.

    CAS  PubMed  Google Scholar 

  9. Briand L, Nespoulous C, Huet JC, Takahashi M, Pernollet JC. Ligand binding and physico-chemical properties of ASP2, a recombinant odorant-binding protein from honeybee (Apis mellifera L.). Eur J Biochem. 2001;268:752–60.

    CAS  PubMed  Google Scholar 

  10. Hulme EC, Birdsall NJM. Strategy and tactics in receptor- binding studies. In: Hulme EC, editor. Receptor–ligand interactions – a practical approach. IRL Press: Oxford; 1992. p. 63–176.

    Google Scholar 

  11. Pang X, Zhou H. Rate constants and mechanisms of protein-ligand binding. Annu Rev Biophys. 2017;46:105–30.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hulme EC, Trevethick MA. Ligand binding assays at equilibrium: validation and interpretation. Br J Pharmacol. 2010;161:1219–37.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Sun YF, De Biasio F, Qiao HL, Iovinella I, Yang SX, Ling Y, et al. Two odorant-binding proteins mediate the behavioural response of aphids to the alarm pheromone (E)-ß-farnesene and structural analogues. PLoS One. 2013;7:e32759.

    Google Scholar 

  14. Leal GM, Leal WS. Binding of a fluorescence reporter and a ligand to an odorant-binding protein of the yellow fever mosquito, Aedes aegypti. F1000 Res. 2015;3:305.

    Google Scholar 

  15. Pelosi P, Baldaccini NE, Pisanelli AM. Identification of a specific olfactory receptor for 2-isobutyl-3-methoxypyrazine. Biochem J. 1982;201:245–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Pelosi P, Iovinella I, Felicioli A, Dani FR. Soluble proteins of chemical communication: an overview across arthropods. Front Physiol. 2014;5:320.

    PubMed  PubMed Central  Google Scholar 

  17. Pelosi P, Iovinella I, Zhu J, Wang G, Dani FR. Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects 2018;93:184–200.

  18. Vogt R.G, Riddiford LM. Pheromone binding and inactivation by moth antennae, Nature 1981;293:161–163.

    CAS  PubMed  Google Scholar 

  19. Bianchet MA, Bains G, Pelosi P, Pevsner J, Snyder SH, Monaco HL, et al. The three dimensional structure of bovine odorant-binding protein and its mechanism of odor recognition. Nat Struct Biol. 1996;3:934–9.

    CAS  PubMed  Google Scholar 

  20. Tegoni M, Ramoni R, Bignetti E, Spinelli S, Cambillau C. Domain swapping creates a third putative combining site in bovine odorant binding protein dimer. Nat Struct Biol. 1996;3:863–7.

    CAS  PubMed  Google Scholar 

  21. Sandler BH, Nikonova L, Leal WS, Clardy J. Sexual attraction in the silkworm moth: structure of the pheromone-binding-protein-bombykol complex. Chem Biol. 2000;7:143–51.

    CAS  PubMed  Google Scholar 

  22. Tegoni M, Campanacci V, Cambillau C. Structural aspects of sexual attraction and chemical communication in insects. Trends Biochem Sci. 2004;29:257–64.

    CAS  PubMed  Google Scholar 

  23. Zhu J, Arena S, Spinelli S, Liu D, Zhang G, Wei R, et al. Reverse chemical ecology: olfactory proteins from the giant panda and their interactions with putative pheromones and bamboo volatiles. Proc Natl Acad Sci U S A. 2017;114:E9802–10.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Dal Monte M, Andreini I, Revoltella R, Pelosi P. Purification and characterization of two odorant binding proteins from nasal tissue of rabbit and pig. Comp Biochem Physiol. 1991;99B:445–51.

    CAS  Google Scholar 

  25. Paolini S, Scaloni A, Amoresano A, Marchese S, Napolitano E, Pelosi P. Amino acid sequence, post-translational modifications, binding and labelling of porcine odorant-binding protein. Chem Senses. 1998;23:689–98.

    CAS  PubMed  Google Scholar 

  26. Spinelli S, Ramoni R, Grolli S, Bonicel J, Cambillau C, Tegoni M. The structure of the monomeric porcine odorant binding protein sheds light on the domain swapping mechanism. Biochemistry. 1998;37:7913–8.

    CAS  PubMed  Google Scholar 

  27. Vincent F, Spinelli S, Ramoni R, Grolli S, Pelosi P, Cambillau C, et al. Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes. J Mol Biol. 2000;300:127–39.

    CAS  PubMed  Google Scholar 

  28. Flower DR, North AC, Sansom CE. The lipocalin protein family: structural and sequence overview. Biochim Biophys Acta. 2000;1482:9–24.

    CAS  PubMed  Google Scholar 

  29. Wei Y, Brandazza A, Pelosi P. Binding of polyclycic aromatic hydrocarbons to mutants of odorant-binding protein: a first step towards biosensors for environmental monitoring. Biochim Biophys Acta. 1784;2008:666–71.

    Google Scholar 

  30. Mulla MY, Tuccori E, Magliulo M, Lattanzi G, Palazzo G, Persaud K, et al. Capacitance-modulated transistor detects odorant binding protein chiral interactions. Nat Commun. 2015;6:6010.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Peitsch M. Protein modeling by E-mail. Bio/Technology. 1995;13:658–60.

    CAS  Google Scholar 

  32. Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics. 2006;22:195–201.

    CAS  PubMed  Google Scholar 

  33. Kiefer F, Arnold K, Künzli M, Bordoli L, Schwede T. The SWISS- MODEL repository and associated resources. Nucleic Acids Res. 2009;37:D387–92.

    CAS  PubMed  Google Scholar 

  34. Grosdidier A, Zoete V, Michielin O. SwissDock, a protein-small molecule docking web service based on EADock DSS. Nucleic Acids Res. 2011;39(Web Server issue):W270–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera. A visualization system for exploratory research and analysis. J Comput Chem 2004;25:1605–1612.

    CAS  PubMed  Google Scholar 

  36. Ban LP, Scaloni A, D’Ambrosio C, Zhang L, Yan YH, Pelosi P. Biochemical characterisation and bacterial expression of an odorant-binding protein from Locusta migratoria. Cell Mol Life Sci. 2003;60:390–400.

    CAS  PubMed  Google Scholar 

  37. Ban LP, Zhang L, Yan YH, Pelosi P. Binding properties of a locust’s chemosensory protein. Biochem Biophys Res Commun. 2002;293:50–4.

    CAS  PubMed  Google Scholar 

  38. Campanacci V, Krieger J, Bette S, Sturgis JN, Lartigue A, Cambillau C, et al. Revisiting the specificity of Mamestra brassicae and Antheraea polyphemus pheromone-binding proteins with a fluorescence binding assay. J Biol Chem. 2001;276:20078–84.

    CAS  PubMed  Google Scholar 

  39. Marie AD, Veggerby C, Robertson DHL, Gaskell SJ, Hubbard SJ, Martinsen L, et al. Effect of polymorphisms on ligand binding by mouse major urinary proteins. Protein Sci. 2001;10:411–7.

    CAS  Google Scholar 

  40. D'Innocenzo B, Salzano AM, D'Ambrosio C, Gazzano A, Niccolini A, Sorce C, et al. Secretory proteins as potential semiochemical carriers in the horse. Biochemistry. 2006;45:13418–28.

    CAS  PubMed  Google Scholar 

  41. Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol. 1965;12:88–118.

    CAS  PubMed  Google Scholar 

  42. Changeux JP. 50 years of allosteric interactions: the twists and turns of the models. Nat Rev Mol Cell Biol. 2013;14:819–29.

    CAS  PubMed  Google Scholar 

  43. Meijer FA, Leijten-van de Gevel IA, de Vries RMJM, Brunsveld L. Allosteric small molecule modulators of nuclear receptors. Mol Cell Endocrinol. 2019;485:20–34.

    CAS  PubMed  Google Scholar 

  44. Changeux JP, Christopoulos A. Allosteric modulation as a unifying mechanism for receptor function and regulation. Diabetes Obes Metab. 2017;19 Suppl 1:4–21.

    CAS  PubMed  Google Scholar 

  45. May LT, Leach K, Sexton PM, Christopoulos A. Allosteric modulation of G protein-coupled receptors. Annu Rev Pharmacol Toxicol. 2007;47:1–51.

    CAS  PubMed  Google Scholar 

  46. Bock A, Schrage R, Mohr K. Allosteric modulators targeting CNS muscarinic receptors. Neuropharmacology. 2018;136:427–37.

    CAS  PubMed  Google Scholar 

  47. Chatzidaki A, Millar NS. Allosteric modulation of nicotinic acetylcholine receptors. Biochem Pharmacol. 2015;97:408–17.

    CAS  PubMed  Google Scholar 

  48. Wingler LM, Elgeti M, Hilger D, Latorraca NR, Lerch MT, Staus DP, et al. Angiotensin analogs with divergent bias stabilize distinct receptor conformations. Cell. 2019;176:468–478.e11.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Patrick JW, Boone CD, Liu W, Conover GM, Liu Y, Cong X, et al. Allostery revealed within lipid binding events to membrane proteins. Proc Natl Acad Sci U S A. 2018;115:2976–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Tegoni M, Pelosi P, Vincent F, Spinelli S, Campanacci V, Grolli S, et al. Mammalian odorant binding proteins. Biochim Biophys Acta. 2000;1482:229–40.

    CAS  PubMed  Google Scholar 

  51. Böcskei Z, Groom CR, Flower DR, Wright CE, Phillips SE, Cavaggioni A, et al. Pheromone binding to two rodent urinary proteins revealed by X-ray crystallography. Nature. 1992;360:186–8.

    PubMed  Google Scholar 

  52. Spinelli S, Vincent F, Pelosi P, Tegoni M, Cambillau C. Boar salivary lipocalin: three-dimensional X-ray structure and androstenol/ androstenone docking simulations. Eur J Biochem. 2002;269:2449–56.

    CAS  PubMed  Google Scholar 

  53. Campanacci V, Lartigue A, Hallberg BM, Jones TA, Giudici-Orticoni MT, Tegoni M, et al. Moth chemosensory protein exhibits drastic conformational changes and cooperativity on ligand binding. Proc Natl Acad Sci U S A. 2003;29:5069–74.

    Google Scholar 

  54. Scaloni A, Monti M, Angeli S, Pelosi P. Structural analysis and disulfide-bridge pairing of two odorant-binding proteins from Bombyx mori. Biochem Biophys Res Commun. 1999;266:386–91.

    CAS  PubMed  Google Scholar 

  55. Leal WS, Nikonova L, Peng G. Disulfide structure of the pheromone binding protein from the silkworm moth Bombyx mori. FEBS Lett. 1999;464:85e90.

    Google Scholar 

Download references

Author information

Author notes

  1. Jiajun Tan and Valeriia Zaremska contributed equally to this work.

Authors and Affiliations

  1. Biosensor Technologies, Austrian Institute of Technology GmbH, Konrad-Lorenz Straße, 24, 3430, Tulln, Austria

    Jiajun Tan, Valeriia Zaremska, Wolfgang Knoll & Paolo Pelosi

  2. School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, Singapore, 637457, Singapore

    Jiajun Tan & Sierin Lim

Authors
  1. Jiajun Tan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Valeriia Zaremska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sierin Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wolfgang Knoll
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Paolo Pelosi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

PP and WK conceived and supervised the study; TJJ and VZ designed and performed experiments; WK provided new tools and reagents; SL and PP analysed data; PP and WK wrote the manuscript; all authors revised and approved the manuscript.

Corresponding author

Correspondence to Paolo Pelosi.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tan, J., Zaremska, V., Lim, S. et al. Probe-dependence of competitive fluorescent ligand binding assays to odorant-binding proteins. Anal Bioanal Chem 412, 547–554 (2020). https://doi.org/10.1007/s00216-019-02309-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-02309-9

Keywords

Search

Navigation