Enhancement, Equal Fluorescence Efficiency, and Quenching in the Interpretation of Fluorescence Anisotropy Data

Abstract

Decreases in fluorescence anisotropy from homo-FRET processes can report on the number of dyes on a protein or the size of a cluster. Fluorescent dyes exhibit a range of behaviours from fluorescence enhancement to quenching when assembled into clusters. The degree of enhancement and/or quenching depends on the number of dyes in close proximity. When interpreting anisotropy, an assumption of equal fluorescence intensity is widely applied. This assumption predicts, for example, that 3 fluorophores in a cluster have the same fluorescence intensity as the same three fluorophores outside of a cluster. This assumption will give incorrect predictions in cases where either quenching or enhancement of fluorescence occurs. Additionally, existing theory typically assumes that all positions within a cluster are equivalent.Application of these assumptions affects interpretation in two ways. It will tend to under-predict the anisotropy of a stochastic mix of individual species and, depending on whether the dye system is quenched or enhanced, will either under-predict or over-predict cluster sizes.Based on computations exploring the impact of enhancement, quenching, and inhomogeneous clustering, a number of conclusions may be reached. As fractional labeling approaches one all models converge to the same value. Inhomogeneous labeling tends to increase anisotropy at low fractional labeling. Applying equal fluorescence intensity assumptions to a fluorescence enhanced system of dyes will over-predict cluster size. Applying it to a quenched system will under-predict cluster size. These cases will be illustrated with simulations and experimental data.