Skip to main content
SpringerLink
Log in

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

  • Article
  • Published:
European Biophysics Journal Aims and scope Submit manuscript

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.

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
Fig. 7

Similar content being viewed by others

References

  • Ausserre D, Hervet H, Rondelez F (1986) Concentration dependence of the interfacial depletion layer thickness for polymer solutions in contact with nonadsorbing walls. Macromolecules 19:85–88

    CAS  Google Scholar 

  • 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 264:381–386

    Article  CAS  Google Scholar 

  • Burghardt TP, Axelrod D (1983) Total internal reflection fluorescence study of energy transfer in surface-adsorbed and dissolved bovine serum albumin. Biochemistry 22:979–985

    CAS  PubMed  Google Scholar 

  • Burghardt TP, Thompson NL (1984) Effect of planar dielectric interfaces on fluorescence emission and detection. Evanescent excitation with high-aperture collection. Biophys J 46:729–737

    CAS  PubMed  Google Scholar 

  • 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 102:10326–10333

    Article  CAS  Google Scholar 

  • 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 20:341–250

    Article  CAS  PubMed  Google Scholar 

  • Crystall B, Rumbles G, Smith TA (1993) Time resolved evanescent wave induced fluorescence measurements of surface adsorbed bovine serum albumin. J Colloid Interface Sci 155:247–250

    Article  CAS  Google Scholar 

  • de Mello AJ, Crystall B, Rumbles G (1995) Evanescent wave spectroscopic studies of surface enhanced fluorescence quantum efficiencies. J Colloid Interface Sci 169:161–167

    Article  Google Scholar 

  • de Mello AJ, Elliott JA, Rumbles G (1997) Evanescent wave-induced fluorescence study of rhodamine 101 at dielectric interfaces. J Chem Soc Faraday Trans 93:4723–4731

    Google Scholar 

  • Edwards J, Ausserre D, Hervet H, Rondelez F (1989) Quantitative studies of evanescent wave intensity profiles using optical fluorescence. Appl Opt 28:1881–1884

    CAS  Google Scholar 

  • Fukumura H, Hayashi K (1990) Time-resolved fluorescence anisotropy of labelled plasma proteins adsorbed on polymer surfaces. J Colloid Interface Sci 135:435–442

    CAS  Google Scholar 

  • Ghiggino KP, Smith TA (1993) Dynamics of energy migration and trapping in photoirradiated polymers. Prog React Kinet 18:375–436

    CAS  Google Scholar 

  • 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 99:4980–4985

    CAS  Google Scholar 

  • Harrick NJ (1967) Internal reflection spectroscopy. Wiley-Interscience, New York

  • Hellen EH, Axelrod D (1987) Fluorescence emission at dielectric and metal-film interfaces. J Opt Soc Am B 4:337–350

    CAS  Google Scholar 

  • Hlady V, Andrade JD (1988) Fluorescence emission from adsorbed bovine serum albumin and albumin-bound 1-anilinonaphthalene-8-sulfonate studied by TIRF. Colloids Surf 32:359–369

    CAS  Google Scholar 

  • Hlady V, Andrade JD (1989) A TIRF titration study of 1-anilinonaphthalene-8-sulfonate binding to silica-adsorbed bovine serum albumin. Colloids Surf 42:85–96

    CAS  Google Scholar 

  • Hlady V, Gölander C, Andrade JD (1988) Hydrophobicity gradient on silica surfaces: a study using total internal reflection fluorescence spectroscopy. Colloids Surf 33:185–190

    CAS  Google Scholar 

  • Lakowicz JR (1991) Topics in fluorescence spectroscopy, vols 1–3. Plenum Press, New York

  • 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 18:9924–9931

    CAS  Google Scholar 

  • 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 62:2083–2092

    CAS  Google Scholar 

  • 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 69:1495–1503

    CAS  Google Scholar 

  • 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–XXXX

    Google Scholar 

  • Lukosz W, Kunz RE (1977b) Light emission by magnetic dipoles close to a plane interface. I. Total radiated power. J Opt Soc Am 67:1607–1614

    Google Scholar 

  • 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 66:2433–2440

    CAS  Google Scholar 

  • Masuhara H, Mataga N, Tazuke S, Murao T, Yamazaki I (1983) Time-resolved total internal reflection fluorescence spectroscopy of polymer films. Chem Phys Lett 100:415–419

    CAS  Google Scholar 

  • O’Connor DV, Phillips D (1984) Time correlated single photon counting. Academic Press, London

  • Parsons D, Harrop R, Mahers EG (1992) The kinetics of particle and polymer adsorption by total internal reflection fluorescence. Colloids Surf 64:151–160

    CAS  Google Scholar 

  • Piasecki DA, Wirth MJ (1994) Spectroscopic probing of the interfacial roughness of sodium dodecyl sulfate adsorbed to a hydrocarbon surface. Langmuir 10:1913–1918

    CAS  Google Scholar 

  • 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 21:539–555

    CAS  Google Scholar 

  • Ratner BD (1989) In: Aggarwal S (ed) Comprehensive polymer science: the synthesis, characterization, reactions and applications of polymers, vol 7. Pergamon Press, Oxford, pp 201–247

  • 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 87:825–830

    CAS  Google Scholar 

  • 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 269–286

  • 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 36–42

  • 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 65

  • 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 276:1032–1037

    CAS  Google Scholar 

  • Suci P, Hlady V (1990) Fluorescence lifetime components of Texas Red-labelled bovine serum albumin: comparison of bulk and adsorbed states. Colloids Surf 51:89–104

    CAS  Google Scholar 

  • 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 257–268

  • 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 12:6717–6720

    Article  CAS  Google Scholar 

  • Wirth MJ (1993) Magic angle lifetime measurements in evanescent wave fluorometry. Appl Spectrosc 47:651–653

    CAS  Google Scholar 

  • Wirth MJ, Burbage JD (1991) Adsorbate reorientation at a water/(octadecylsilyl)silica interface. Anal Chem 63:1311–1317

    CAS  Google Scholar 

  • 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 102:7691–7694

    CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. School of Chemistry, University of Melbourne, 3010, Parkville, Victoria , Australia

    Michelle L. Gee, Levie Lensun, Trevor A. Smith & Colin A. Scholes

Authors
  1. Michelle L. Gee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Levie Lensun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Trevor A. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Colin A. Scholes
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michelle L. Gee.

Additional information

Submitted as a record of the 2002 Australian Biophysical Society meeting

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gee, M.L., Lensun, L., Smith, T.A. et al. Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface. Eur Biophys J 33, 130–139 (2004). https://doi.org/10.1007/s00249-003-0360-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00249-003-0360-5

Keywords

Search

Navigation