Stern-Volmer Modeling of Steady-State Forster Energy Transfer Between Dilute, Freely Diffusing Membrane-Bound Fluorophores

TL;DR

This paper demonstrates that simple Stern-Volmer models effectively describe steady-state FRET between membrane-bound fluorophores under dilute conditions, linking experimental data with theoretical models for better experiment design.

Contribution

It introduces a validated Stern-Volmer modeling approach for membrane FRET and establishes a novel link with Wolber and Hudson's quenching approximation.

Findings

Stern-Volmer expressions accurately model FRET metrics in dilute membrane conditions

A new correspondence links Stern-Volmer constants to Forster distances and concentration limits

Provides a three-step strategy for designing effective FRET experiments in biomembranes

Abstract

Two different metrics are used to assess Forster resonance energy transfer (FRET) between fluorophores in the steady state: (1) acceptor-quenching of donor fluorescence, E (a.k.a. transfer efficiency); and (ii) donor-excited acceptor fluorescence, F-A-Dex. While E is still more widely used, F-A-Dex has been gaining in popularity for practical reasons among experimentalists who study biomembranes. Here, for the special case of membrane-bound fluorophores, we present a substantial body of experimental evidence that justifies the use of simple Stern-Volmer expressions when modeling either FRET metric under dilute-probe conditions. We have also discovered a dilute-regime correspondence between our Stern-Volmer expression for E and Wolber and Hudson's series approximation for steady-state Forster quenching in 2D. This novel correspondence allows us to interpret each of our 2D quenching constants in terms of both (i) an effective Forster distance, and (ii) two maximum acceptor-concentration limits, each of which defines its own useful experimental regime. Taken together, our results suggest a three-step strategy toward designing more effective steady-state FRET experiments for the study of biomembranes.