Structural changes of fibronectin adsorbed to model surfaces probed by fluorescence resonance energy transfer.
Structural changes of proteins during adsorption to biomaterials affect the presentation of molecular binding sites and, ultimately, biomaterial performance. We have applied fluorescence resonance energy transfer (FRET) spectroscopy to study structural changes of the cell adhesion protein, fibronectin (Fn), following adsorption to model hydrophilic and hydrophobic surfaces. Fn was labeled with donor and acceptor fluorophores using two labeling schemes and intramolecular energy transfer was calibrated against measured structural changes of Fn in denaturing solutions. FRET was then applied to measure Fn's structure on surfaces. Based on FRET, Fn underwent greater extension of its dimer arms on hydrophilic glass than on hydrophobic fluoroalklysilane-derivatized glass (fluorosilane), and this extension was insensitive to molecular packing over a range of adsorption concentrations. Fn's conformation on glass better promoted cell attachment than on fluorosilane; the roles of both global structural changes (movements of modules) and local structural changes (disruption of secondary structure) on Fn's cell integrin binding activity are discussed. Based on previous FRET work, we compare Fn's conformations on these surfaces with its conformations in fibroblast culture. FRET is unique in allowing direct comparison of protein structure between biomaterial surfaces and cell culture.
Baugh
Loren
L
Center for Nanotechnology and Department of Bioengineering, University of Washington, Box 351721, Seattle, Washington 98195, USA.
Vogel
Viola
V
D000327:Adsorption; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry* / Q000378:metabolism; D002448:Cell Adhesion; D005353:Fibronectins / Q000737:chemistry* / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D005459:Fluorides / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D019791:Guanidine / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells / Q000166:cytology / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D012821:Silanes / Q000737:chemistry; D013499:Surface Properties
D001672:Biocompatible Materials; D005353:Fibronectins; D005456:Fluorescent Dyes; D012821:Silanes; D019791:Guanidine; D005459:Fluorides