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

Eur Biophys J. 2004 Apr;33(2):130-9. doi: 10.1007/s00249-003-0360-5. Epub 2003 Oct 28.

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adsorption
  • Algorithms*
  • Equipment Design
  • Fluorescence Polarization / instrumentation
  • Fluorescence Polarization / methods*
  • Protein Conformation
  • Serum Albumin, Bovine / analysis
  • Serum Albumin, Bovine / chemistry*
  • Silicon Dioxide / chemistry
  • Spectrometry, Fluorescence / instrumentation
  • Spectrometry, Fluorescence / methods*
  • Surface Properties
  • Water / chemistry*

Substances

  • Water
  • Serum Albumin, Bovine
  • Silicon Dioxide