ExiFRET: flexible tool for understanding FRET in complex geometries

Abstract

Abstract. Fluorescence resonance energy transfer (FRET) can be utilized to gain low-resolution structural information by reporting on the proximity of molecules or measuring inter-and intramolecular distances. This method exploits the fact that the probability of the energy transfer is related to the separation between the fluorescent molecules. This relationship is well described for a single pair of fluorophores but is complicated in systems containing more than two fluorophores. Here, we present a Monte Carlo calculation scheme that has been implemented through a user-friendly web-based program called ExiFRET that can be used to determine the FRET efficiency in a wide range of fluorophore arrangements. ExiFRET is useful to model FRET for individual fluorophores randomly distributed in two or three dimensions, fluorophores linked in pairs or arranged in regular geometries with or without predefined stoichiometries. ExiFRET can model both uniform distributions and fluorophores that are aggregated in clusters. We demonstrate how this tool can be employed to understand the effect of labeling efficiency on FRET efficiency, estimate relative contributions of inter-and intramolecular FRET, investigate the structure of multimeric proteins, stoichiometries, and oligomers, and to aid experiments studying the aggregation of lipids and proteins in membrane environments. We also present an extension that can be used to study instances in which fluorophores have constrained orientations.

Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications

“…345,346 This latter aspect is important because high excitation intensities may lead to many excited Ds and/or As (directly excited and/or via FRET by the multiple available Ds), and as a consequence the already excited As will be unavailable for FRET and E FRET will decrease. This means that eq 18 is valid only for low excitation intensities, for which all Ds and As have decayed back to their ground states before the next photon arrives for excitation.…”

Förster Resonance Energy Transfer (FRET) Correlates of Altered Subunit Stoichiometry in Cys-Loop Receptors, Exemplified by Nicotinic α4β2

“…We show that the new method does not require additional strong assumptions about symmetry, or about distances between fluorophores. The method may be considered a special case within the tradition of analyzing pentameric proteins by FRET [20–23]. The theory is of interest because the measurements have yielded estimates about changes in stoichiometry that are consistent with known structural information and with chaperoning by nicotine, as well as a novel effect of chaperoning by cytisine.…”

Lateral diffusion contributes to FRET from lanthanide-tagged membrane proteins

“…This assumption maximizes the estimated FRET efficiency, and is consistent with exposure of covalently-linked benzyl-linker-fluorophore substrates on the surface of the alkyltransferase SNAP-tag (PDB: 3L00). Monte Carlo simulations were carried out using the same parameters and ExiFRET (www.exifret.com) [6]. …”