Förster Resonance Energy Transfer (FRET) between Heterogeneously Distributed Probes: Application to Lipid Nanodomains and Pores

Radek Šachl,Martin Hof,Lennart Johansson

doi:10.3390/ijms131216141

Abstract

The formation of membrane heterogeneities, e.g., lipid domains and pores, leads to a redistribution of donor (D) and acceptor (A) molecules according to their affinity to the structures formed and the remaining bilayer. If such changes sufficiently influence the Förster resonance energy transfer (FRET) efficiency, these changes can be further analyzed in terms of nanodomain/pore size. This paper is a continuation of previous work on this theme. In particular, it is demonstrated how FRET experiments should be planned and how data should be analyzed in order to achieve the best possible resolution. The limiting resolution of domains and pores are discussed simultaneously, in order to enable direct comparison. It appears that choice of suitable donor/acceptor pairs is the most crucial step in the design of experiments. For instance, it is recommended to use DA pairs, which exhibit an increased affinity to pores (i.e., partition coefficients KD,A > 10) for the determination of pore sizes with radii comparable to the Förster radius R0. On the other hand, donors and acceptors exhibiting a high affinity to different phases are better suited for the determination of domain sizes. The experimental setup where donors and acceptors are excluded from the domains/pores should be avoided.

Highlights

IntroductionLiquid ordered Lo domains (referred to as rafts) [1] and pores [2] represent heterogeneities usually encountered in lipid bilayers
Liquid ordered Lo domains [1] and pores [2] represent heterogeneities usually encountered in lipid bilayers
It can be expected that the formation of domains/pores in the lipid bilayer disturbs a homogenous distribution of fluorescent probes

Summary

Introduction

Liquid ordered Lo domains (referred to as rafts) [1] and pores [2] represent heterogeneities usually encountered in lipid bilayers. The situation can be more favorable in the case of pores, which are usually built by bigger (as compared to the size of a fluorescent dye) protein/peptide molecules. In a typical qualitative FRET experiment, the FRET efficiencies of a DA pair localized in a bilayer, which does and does not contain membrane heterogeneities, are compared This should preferentially be done by comparison of time-resolved fluorescence (TRF) decays for the two situations, but measurement of steady-state (SS) intensities is in principle possible as well. A certain peptide fraction is labeled by a fluorescent donor (green balls) in the polar end part This means that donors are localized in the pores at the lipid/water interface only (KD = ∞). In order to highlight the pore-structure in the illustration, the pores have been heavily stained by the fluorophores

Limitations of FRET in the Detection of Nanodomains and Cylindrical Pores

Searching for the Size of the Liquid-Ordered Domains

FRET Modeling of Uniformly Distributed Probes

Handling the Lack of Analytical Models for Heterogeneous Probe Distributions

Monte Carlo Simulations

Conclusions