The investigation of biomolecules in solution is key to gathering information about their conformational states, transitions, dynamics, interactions and functional mechanisms. Förster resonance energy transfer (FRET) is integral in elucidating these parameters as it can be used as a molecular ruler at the biomolecular level. 

First single molecule fluorescence experiments on single enzymes in droplets were performed by Rotman in 1961 [1] and on single molecules with 80-90 fluorophores by Hirschfeld in 1976 [2]. In 1990, Brooks Shera et al. described the detection of individual fluorophores [3], and single-pair Förster resonance energy transfer was first mentioned by Ha et al. (1996) [4], Deniz et al. (1999) [5], and Weiss (1999) [6]. 

Förster resonance energy transfer is the non-radiative transfer of energy from a donor fluorophore in its excited state to an acceptor in close proximity by dipole-dipole interactions. The value of the energy transfer is determined by the spectral overlap and the distance between the donor and the acceptor fluorophores. It can be used to obtain distance and structural information about molecules and their dynamics in a distance range of 20-80 Å [7], [8]. Thus, FRET can be used as an ideal tool to measure intramolecular and intermolecular distances. Therefore, it allows for the investigation of conformational states and changes, protein-nucleic acid, protein-protein, and protein-lipid interactions in vitro and in cellulo.

Here, we feature a methods collection of articles around Förster resonance energy transfer. The collection provides articles on ensemble and single molecule two- and multicolor FRET ranging from in vitro studies in liquids, in lipid bilayers or in three dimensional hydrogels [9] to live cell measurements. Single molecule FRET was used to obtain kinetic information by Hidden Markov Models and to understand the dynamics and interactions of complex protein systems [10]. For example, active and inactive states of AURKA kinase [11], the enzyme kinetics of SUMO/Sentrin specific protease 1 (SENP1) [12], Ca2+-ATPase structure and function [13], and the dissociation of Holliday junctions were investigated [14]. By time-resolved single molecule protein-induced fluorescence enhancement, the local confirmations of α-synuclein were analyzed [15]. Furthermore, FRET was used to measure the binding kinetics of T-cell antigen receptor and MHC molecules in situ [16].

Multiple articles of the methods collection describe the use of FRET in living cells. FRET-based tension sensors were used, for example, in combination with fluorescence recovery after photobleaching to analyze the focal adhesion protein vinculin inside living cells [17]. Furthermore, a combination of FRET and fluorescence cross-correlation spectroscopy was applied to investigate membrane receptor dynamics [18] and phosphorylated STAT proteins were followed by time-resolved FRET in cells [19]. Finally, Schmidtpott and Seidel (2021) describe the application of FRET to living plant cells [20]. 

In the collection, we learn about details on the selection of sites for labeling, the choice of fluorophores, acquisition and data analysis for FRET mapping [21]. An article by Kim et al. (2016) explains the use of a portable analyzer for FRET sensor data for maltose detection [22] and Schrangl et al. (2021) provide a software toolkit for the spatiotemporal analysis of smFRET time traces [23].

The wide range of tools, analysis approaches, and applications of FRET detailed in this collection provide an overview of the wide range of the use of FRET in the investigation of biomolecular processes and beyond.