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.
Gee
Michelle L
ML
School of Chemistry, University of Melbourne, 3010 Parkville, Victoria, Australia. mlgee@unimelb.edu.au
Lensun
Levie
L
Smith
Trevor A
TA
Scholes
Colin A
CA
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