Cooperativity in the Binding of Avidin to Biotin-Lipid-Doped Langmuir-Blodgett Films

Monolayers of arachidic acid (AA) doped with either biotinylated DPPE (B-DPPE) or a chain extended biotinylated DPPE (B-x-DPPE) were deposited onto alkylsilane treated surfaces of quartz evanescent fiber optic sensors (EFO) by the Langmuir-Blodgett (LB) technique. The surfacemodified EFOs were usedto obtain binding isotherms of fluorescein-labeled avidinto the biotin-lipid-doped LB fii. Hyperbolic binding isotherms were observed for all B-DPPE doped LB films and for B-x-DPPE doped fiis with <0.63 mol 76 biotin lipid. Sigmoid or positively cooperative binding isotherm were observed for all LB Tis with 20.63 mol 76 B-x-DPPE. A mathematical expression for protein binding to a two-dimensional array of receptors that takes protein-protein interaction into account was used to quantitatively assess the cooperativity observed in the isotherms. Attenuated total reflection Fourier transform infrared (ATRFTIR) spectroscopy was used to address speculation that cooperativity resulted from a conformational change in avidin. ATR-FTIR results show that avidin experienced significant conformational changes when bound to biotin lipids in the LB fiis, wheras no conformational change was observed for avidin nonspecifically bound to biotin-free LB films.