Time-resolved evanescent wave-induced fluorescence anisotropy for the determination of molecular conformational changes of proteins at an interface.
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.
School of Chemistry, University of Melbourne, 3010 Parkville, Victoria, Australia. mlgee@unimelb.edu.au
Michelle L
ML
Gee
Levie
L
Lensun
Trevor A
TA
Smith
Colin A
CA
Scholes
D000327:Adsorption; D000465:Algorithms; D004867:Equipment Design; D005454:Fluorescence Polarization / Q000295:instrumentation / Q000379:methods*; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*; D013499:Surface Properties; D014867:Water / Q000737:chemistry*
D014867:Water; D012710:Serum Albumin, Bovine; D012822:Silicon Dioxide