Analysis of vertical fluorescence resonance energy transfer from the surface of a small-diameter sphere

Fluorescence resonance energy transfer (FRET) measurements have been used t o analyze fluorophore separations in a number of varying geometries, includ ing small particles and extended surfaces. This study focuses on the geomet ry created by a donor extended above the surface of a small sphere (radius < R-0), where the accepters are integrated into the sphere surface. The mod el of this geometry was based on an amphipathic molecule with its lipophili c region integrated into a detergent micelle and its hydrophilic region ext ending outward from the micelle surface, where the donor fluorophore is att ached to the hydrophilic region of the molecule. Based on random acceptor i ncorporation into the micelle, a Poisson distribution was used to calculate the distribution of acceptor probes across the micelle population. The mod el converges to RET on a flat surface when the radius of the micelle exceed s 0.8 R-0. The model was also used to simulate FRET data showing that the p ositions of donors above the micelle surface could be uniquely resolved. Ex perimental verification of the model was achieved in a sulfobetaine palmita te micelle with fluorescein isothiocyanate donors attached to detergent-sol ubilized lipopolysaccharide (LPS) and lipophilic Fast-Dir accepters. The us e of steady-state analysis allowed resolution of cases in which donors were located at different distances from the surface. Combining steady-state wi th excited-state lifetime analysis allowed resolution of cases where there was a combination of distances. Given the large number of biomolecules that interact with lipids, this approach may prove generally useful for definin g molecular conformation.