European Biophysics Journal

, Volume 33, Issue 2, pp 130–139 | Cite as

Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface

  • Michelle L. Gee
  • Levie Lensun
  • Trevor A. Smith
  • Colin A. Scholes
Article

Abstract

We have shown that the molecular conformation of a protein at an interface can be probed spatially using time-resolved evanescent wave-induced fluorescence spectroscopic (TREWIFS) techniques. Specifically, by varying the penetration depth of the evanescent field, variable-angle TREWIFS, coupled with variable-angle evanescent wave-induced time-resolved fluorescence anisotropy measurements, allow us to monitor how fluorescence intensity and fluorescence depolarization vary normal to an interface as a function of time after excitation. We have applied this technique to the study of bovine serum albumin (BSA) complexed noncovalently with the fluorophore 1-anilinonaphthalene-8-sulfonic acid. The fluorescence decay varies as a function of the penetration depth of the evanescent wave in a manner that indicates a gradient of hydrophobicity through the adsorbed protein, normal to the interface. Restriction of the fluorescent probe’s motion also occurs as a function of distance normal to the interface. The results are consistent with a model of partial protein denaturation: at the surface, an adsorbed BSA molecule unfolds, thus optimizing protein–silica interactions and the number of points of attachment to the surface. Further away, normal to the surface, the protein molecule maintains its coiled structure.

Keywords

Bovine serum albumin Fluorescence depolarization Fluorescence intensity Protein conformation Time-resolved evanescent wave-induced fluorescence spectroscopy 

Notes

Acknowledgements

The authors would like to thank Professor Ken Ghiggino for providing access to the laser equipment. We also acknowledge the Australian Research Council for their generous financial support of the work in the form of an ARC Large Grant.

References

  1. Ausserre D, Hervet H, Rondelez F (1986) Concentration dependence of the interfacial depletion layer thickness for polymer solutions in contact with nonadsorbing walls. Macromolecules 19:85–88Google Scholar
  2. Bessho K, Uchida T, Yamauchi A, Shioya T, Teramae N (1997) Microenvironments of 8-anilino-1-naphthalenesulfonate at the heptane-water interface: time-resolved total internal reflection fluorescence spectroscopy. Chem Phys Lett 264:381–386CrossRefGoogle Scholar
  3. Burghardt TP, Axelrod D (1983) Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin. Biochemistry 22:979–985PubMedGoogle Scholar
  4. Burghardt TP, Thompson NL (1984) Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection. Biophys J 46:729–737PubMedGoogle Scholar
  5. Byrne CD, de Mello AJ, Barnes WL (1998) Variable-angle time-resolved evanescent wave-induced fluorescence spectroscopy (VATR-EWIFS): a technique for concentration profiling fluorophores at dielectric interfaces. J Phys Chem B 102:10326–10333CrossRefGoogle Scholar
  6. Cornelius RM, Brash JL (1999) Adsorption from plasma and buffer of single- and two-chain high molecular weight kininogen to glass and sulphonated polyurethane surfaces. Biomaterials 20:341–250CrossRefPubMedGoogle Scholar
  7. Crystall B, Rumbles G, Smith TA (1993) Time resolved evanescent wave induced fluorescence measurements of surface adsorbed bovine serum albumin. J Colloid Interface Sci 155:247–250CrossRefGoogle Scholar
  8. de Mello AJ, Crystall B, Rumbles G (1995) Evanescent wave spectroscopic studies of surface enhanced fluorescence quantum efficiencies. J Colloid Interface Sci 169:161–167CrossRefGoogle Scholar
  9. de Mello AJ, Elliott JA, Rumbles G (1997) Evanescent wave-induced fluorescence study of rhodamine 101 at dielectric interfaces. J Chem Soc Faraday Trans 93:4723–4731Google Scholar
  10. Edwards J, Ausserre D, Hervet H, Rondelez F (1989) Quantitative studies of evanescent wave intensity profiles using optical fluorescence. Appl Opt 28:1881–1884Google Scholar
  11. Fukumura H, Hayashi K (1990) Time-resolved fluorescence anisotropy of labelled plasma proteins adsorbed on polymer surfaces. J Colloid Interface Sci 135:435–442Google Scholar
  12. Ghiggino KP, Smith TA (1993) Dynamics of energy migration and trapping in photoirradiated polymers. Prog React Kinet 18:375–436Google Scholar
  13. Hamai S, Tamai N, Masuhara H (1995) Excimer formation of pyrene in a solid/polymer solution interface layer. A time-resolved total internal reflection fluorescence study. J Phys Chem 99:4980–4985Google Scholar
  14. Harrick NJ (1967) Internal reflection spectroscopy. Wiley-Interscience, New YorkGoogle Scholar
  15. Hellen EH, Axelrod D (1987) Fluorescence emission at dielectric and metal-film interfaces. J Opt Soc Am B 4:337–350Google Scholar
  16. Hlady V, Andrade JD (1988) Fluorescence emission from adsorbed bovine serum albumin and albumin-bound 1-anilinonaphthalene-8-sulfonate studied by TIRF. Colloids Surf 32:359–369Google Scholar
  17. Hlady V, Andrade JD (1989) A TIRF titration study of 1-anilinonaphthalene-8-sulfonate binding to silica-adsorbed bovine serum albumin. Colloids Surf 42:85–96Google Scholar
  18. Hlady V, Gölander C, Andrade JD (1988) Hydrophobicity gradient on silica surfaces: a study using total internal reflection fluorescence spectroscopy. Colloids Surf 33:185–190Google Scholar
  19. Lakowicz JR (1991) Topics in fluorescence spectroscopy, vols 1–3. Plenum Press, New YorkGoogle Scholar
  20. Lensun L, Smith TA, Gee ML (2002) The partial denaturation of silica-adsorbed bovine serum albumin determined by time-resolved evanescent wave-induced fluorescence spectroscopy. Langmuir 18:9924–9931Google Scholar
  21. Liebmann LW, Robinson JA, Mann KG (1991) A dual beam total internal reflection fluorescence spectrometer for dynamic depth resolved measurements of biochemical liquid-solid Interface binding reactions in opaque solvents. Rev Sci Instrum 62:2083–2092Google Scholar
  22. Lukosz W (1979) Light emission by magnetic and electric dipoles close to a plane dielectric interface. III. Radiation patterns of dipoles with arbitrary orientation. J Opt Soc Am 69:1495–1503Google Scholar
  23. Lukosz W, Kunz RE (1977a) Light emission by magnetic dipoles close to a plane interface. I. Radiation patterns of perpendicular oriented dipoles. J Opt Soc Am 67:1615–XXXXGoogle Scholar
  24. Lukosz W, Kunz RE (1977b) Light emission by magnetic dipoles close to a plane interface. I. Total radiated power. J Opt Soc Am 67:1607–1614Google Scholar
  25. Lundgren JS, Bekos EJ, Wang R, Bright FV (1994) Phase-resolved evanescent wave induced fluorescence. An in situ tool for studying heterogeneous interfaces. Anal Chem 66:2433–2440Google Scholar
  26. Masuhara H, Mataga N, Tazuke S, Murao T, Yamazaki I (1983) Time-resolved total internal reflection fluorescence spectroscopy of polymer films. Chem Phys Lett 100:415–419Google Scholar
  27. O’Connor DV, Phillips D (1984) Time correlated single photon counting. Academic Press, LondonGoogle Scholar
  28. Parsons D, Harrop R, Mahers EG (1992) The kinetics of particle and polymer adsorption by total internal reflection fluorescence. Colloids Surf 64:151–160Google Scholar
  29. Piasecki DA, Wirth MJ (1994) Spectroscopic probing of the interfacial roughness of sodium dodecyl sulfate adsorbed to a hydrocarbon surface. Langmuir 10:1913–1918Google Scholar
  30. Rainbow MR, Atherton S, Eberhart RC (1987) Fluorescence lifetime measurements using total internal reflection fluorimetry: evidence for a conformational change in albumin adsorbed to quartz. J Biomed Mat Res 21:539–555Google Scholar
  31. Ratner BD (1989) In: Aggarwal S (ed) Comprehensive polymer science: the synthesis, characterization, reactions and applications of polymers, vol 7. Pergamon Press, Oxford, pp 201–247Google Scholar
  32. Rumbles G, Brown AJ, Phillips D (1991) Time-resolved evanescent wave induced fluorescence spectroscopy, part 1. Deviations in the fluorescence lifetime of tetrasulphonated aluminium phthalocyanine at a fused silica/methanol interface. J Chem Soc Faraday Trans 87:825–830Google Scholar
  33. Rumbles G, Bloor D, Brown AJ, de Mello AJ, Crystall B, Phillips D, Smith TA (1994) Time-resolved evanescent wave induced fluorescence studies of polymer-surface interactions. In: Masuhara H, Schryver FCD, Kitamura N, Tamai N (eds) Microchemistry: spectroscopy and chemistry in small domains. Elsevier, London, pp 269–286Google Scholar
  34. Schneckenburger H, Stock K, Eickholz J, Strauss WSL, Lyttek M, Sailer R (2000) Time-resolved total internal reflection fluorescence spectroscopy: application to the membrane marker laurdan. In: König K, Tanke HJ, Schneckenburger H (eds) Proc SPIE, laser microscopy, vol 4164. SPIE, Amsterdam, Netherlands, pp 36–42Google Scholar
  35. Schneckenburger H, Sailer R, Stock K, Lyttek M, Strauss WSL (2001) Total internal reflection fluorescence lifetime imaging (TIR-FLIM) of living cells. In: Gu M (ed) Multidimensional microscopy 2001, 3rd Asia–Pacific international symposium on confocal microscopy and related technologies, Melbourne, Australia, p 65Google Scholar
  36. Smith TA, Irwanto M, Haines DJ, Ghiggino KP, Millar DP (1998) Time-resolved fluorescence anisotropy measurements of the adsorption of rhodamine-B and a labelled polyelectrolyte onto colloidal silica. Colloid Polym Sci 276:1032–1037Google Scholar
  37. Suci P, Hlady V (1990) Fluorescence lifetime components of Texas Red-labelled bovine serum albumin: comparison of bulk and adsorbed states. Colloids Surf 51:89–104Google Scholar
  38. Toriumi M, Yanagimachi M (1994) Time-resolved total-internal reflection fluorescence spectroscopy and its applications to solid/polymer interface layers. In: Masuhara H, Schryver FCD, Kitamura N, Tamai N (eds) Microchemistry: spectroscopy and chemistry in small domains. Elsevier, London, pp 257–268Google Scholar
  39. Watarai H, Funaki F (1996) Total internal reflection fluorescence measurements of protonation equilibria of rhodamine B and octadecylrhodamine B at a toluene/water interface. Langmuir 12:6717–6720CrossRefGoogle Scholar
  40. Wirth MJ (1993) Magic angle lifetime measurements in evanescent wave fluorometry. Appl Spectrosc 47:651–653Google Scholar
  41. Wirth MJ, Burbage JD (1991) Adsorbate reorientation at a water/(octadecylsilyl)silica interface. Anal Chem 63:1311–1317Google Scholar
  42. Yao H, Ikeda H, Kitamura N (1998) Surface-induced aggregation of pseudoisocyanine dye at a glass/solution interface studied by total-internal reflection fluorescence spectroscopy. J Phys Chem 102:7691–7694Google Scholar

Copyright information

© EBSA 2004

Authors and Affiliations

  • Michelle L. Gee
    • 1
  • Levie Lensun
    • 1
  • Trevor A. Smith
    • 1
  • Colin A. Scholes
    • 1
  1. 1.School of ChemistryUniversity of MelbourneParkvilleAustralia

Personalised recommendations