Analysis of Vertical Fluorescence Resonance Energy Transfer from the Surface of a Small-Diameter Sphere

Abstract

Fluorescence resonance energy transfer (FRET) measurements have been used to analyze fluorophore separations in a number of varying geometries, including small particles and extended surfaces. This study focuses on the geometry created by a donor extended above the surface of a small sphere (radius < R0), where the acceptors are integrated into the sphere surface. The model of this geometry was based on an amphipathic molecule with its lipophilic region integrated into a detergent micelle and its hydrophilic region extending outward from the micelle surface, where the donor fluorophore is attached to the hydrophilic region of the molecule. Based on random acceptor incorporation into the micelle, a Poisson distribution was used to calculate the distribution of acceptor probes across the micelle population. The model converges to RET on a flat surface when the radius of the micelle exceeds 0.8 R0. The model was also used to simulate FRET data showing that the positions of donors above the micelle surface could be uniquely resolved. Experimental verification of the model was achieved in a sulfobetaine palmitate micelle with fluorescein isothiocyanate donors attached to detergent-solubilized lipopolysaccharide (LPS) and lipophilic Fast-DiI acceptors. The use of steady-state analysis allowed resolution of cases in which donors were located at different distances from the surface. Combining steady-state with 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 defining molecular conformation.

Ultrasensitive Protein Concentration Measurement Based on Particle Adsorption and Fluorescence Quenching

“…The linear relationship between the logarithms of absorption coefficient and the particle diameter was found suggesting that the particle concentrations were correctly provided . According to Jones et al, the total surface area of the differently sized particles should be kept constant to yield the same surface coverage and quenching . However, we observed different quenching efficacies for the particles.…”

“…35 According to Jones et al, the total surface area of the differently sized particles should be kept constant to yield the same surface coverage and quenching. 36 However, we observed different quenching efficacies for the particles. Thus, we selected a number of particles for each particle size giving equal background signal (no BSA sample in the assay) to the comparative study.…”

Mapping the Antagonist Binding Site of the Serotonin Type 3 Receptor by Fluorescence Resonance Energy Transfer

“…To evaluate the FRET efficiency between a pointlike donor and acceptor molecules distributed over a two-dimensional surface, averaging over every allowed configuration and over the statistical distribution of the number of acceptors per micelle is required. We essentially adapted the calculations of Jones et al (18) to our model and refer the reader to this publication and to Wolber et al (19) for further details.…”