ABSTRACT
Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Nucleic Acid Conformation , Protein Conformation , Single Molecule Imaging/methods , Fluorescence Resonance Energy Transfer/history , History, 20th Century , History, 21st Century , Molecular Biology/trends , Single Molecule Imaging/historyABSTRACT
Since its first demonstration about twenty years ago, single-molecule fluorescence resonance energy transfer (FRET) has undergone remarkable technical advances. In this tutorial review, we will discuss two technical advances that increase the information content of the single-molecule FRET measurements: single-molecule multi-color FRET and single-molecule FRET combined with force or torque. Our expectations for future developments will be briefly discussed at the end.
Subject(s)
Fluorescence Resonance Energy Transfer/instrumentation , Fluorescence Resonance Energy Transfer/methods , Equipment Design , Fluorescence Resonance Energy Transfer/history , History, 20th Century , History, 21st Century , Magnetics/instrumentation , Microscopy, Confocal/instrumentation , Microscopy, Confocal/methods , Microscopy, Interference/instrumentation , Microscopy, Interference/methods , Optical TweezersABSTRACT
In the last 10 years, imaging assays based on resonance energy transfer (RET) and protein fragment complementation have made it possible to study interactions between components of G protein-coupled receptor (GPCR) signalling complexes in living cells under physiological conditions. Here, we consider the history of such approaches, the current tools available and how they have changed our understanding of GPCR signalling. We also discuss some theoretical and methodological issues important when combining the different types of assay.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Protein Interaction Maps , Receptors, G-Protein-Coupled/metabolism , Animals , Fluorescence Resonance Energy Transfer/history , History, 20th Century , History, 21st Century , Humans , Signal TransductionABSTRACT
Theodor Förster would have been 100 years old this year, and he would have been astounded to see the impact of his scientific achievement, which is still evolving. Combining his quantitative approach of (Förster) resonance energy transfer (FRET) with state-of-the-art digital imaging techniques allows scientists to breach the resolution limits of light (ca. 200 nm) in light microscopy. The ability to deduce molecular or particle distances within a range of 1-10 nm in real time and to prove or disprove interactions between two or more components is of vital interest to researchers in many branches of science. While Förster's groundbreaking theory was published in the 1940s, the availability of suitable fluorophores, instruments, and analytical tools spawned numerous experiments in the last 20 years, as demonstrated by the exponential increase in publications. These cover basic investigation of cellular processes and the ability to investigate them when they go awry in pathological states, the dynamics involved in genetics, and following events in environmental sciences and methods in drug screening. This review covers the essentials of Theodor Förster's theory, describes the elements for successful implementation of FRET microscopy, the challenges and how to overcome them, and a leading-edge example of how Förster's scientific impact is still evolving in many directions. While this review cannot possibly do justice to the burgeoning field of FRET microscopy, a few interesting applications such as threecolor FRET, which greatly expands the opportunities for investigating interactions of cellular components compared with the traditional two-color method, are described, and an extensive list of references is provided for the interested reader to access.