Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer

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Science  19 Jan 2018:
Vol. 359, Issue 6373, eaan1133
DOI: 10.1126/science.aan1133

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Watching single molecules in motion

Structural techniques such as x-ray crystallography and electron microscopy give insight into how macromolecules function by providing snapshots of different conformational states. Function also depends on the path between those states, but to see that path involves watching single molecules move. This became possible with the advent of single-molecule Förster resonance energy transfer (smFRET), which was first implemented in 1996. Lerner et al. review how smFRET has been used to study macromolecules in action, providing mechanistic insights into processes such as DNA repair, transcription, and translation. They also describe current limitations of the approach and suggest how future developments may expand the applications of smFRET.

Science, this issue p. eaan1133

Structured Abstract


Biomolecular mechanisms are typically inferred from static structural “snapshots” obtained by x-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo–electron microscopy (cryo-EM). In these approaches, mechanisms have to be validated using additional information from established biochemical and biophysical assays. However, linking conformational states to biochemical function requires the ability to resolve structural dynamics, as macromolecular structure can be intrinsically dynamic or altered upon ligand binding. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying such structural dynamics under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments both confirmed previous hypotheses and discovered new fundamental biological mechanisms relevant for DNA maintenance, replication and transcription, translation, protein folding, enzymatic function, and membrane transport. We review the evolution of smFRET as a key tool for “dynamic structural biology” over the past 22 years and highlight the prospects for its use in applications such as biosensing, high-throughput screening, and molecular diagnostics.


FRET was first identified in the 1920s by Cario, Franck, and Perrin. In the late 1940s, Förster and Oppenheimer independently formulated a quantitative theory of the energy transfer between a pair of point dipoles. Stryer and Haugland verified this theory in the late 1960s and coined the term “spectroscopic ruler” for FRET. Simultaneously, Hirschfeld, and later Moerner and Orrit, pioneered optical single-molecule detection methods leading to the first demonstration of smFRET in 1996. This breakthrough made it possible to study heterogeneous systems, dynamic processes, and transient conformational changes on the nanometer scale. The smFRET technique was rapidly adopted by various research groups to provide mechanistic answers in diverse areas of biological research. In early pioneering applications of smFRET in biochemistry, Ha et al. visualized the conformational dynamics of the staphylococcal nuclease enzyme; Deniz et al. obtained information on the structural dynamics of double-stranded DNA; and Zhuang et al. studied the conformation of individual RNA enzyme molecules and their folding dynamics in equilibrium. These pioneering studies were followed by others that used smFRET to unravel the inner workings of helicases and topoisomerases, DNA replication, DNA repair, transcription, translation, enzymatic reactions, molecular motors, membrane proteins, nucleic acids, protein and RNA folding, ribozyme catalysis, and many other molecular mechanisms.


During the past two decades, smFRET has grown into a mature toolset with capabilities to explore dynamic structural biology for both equilibrium and non-equilibrium reactions. The one-dimensional (“ruler”) character of the FRET approach, however, only captures the complex three-dimensional structure of a system and needs to be complemented by other techniques that can provide additional information about the respective biochemical states of macromolecules. Approaches that explore smFRET combinations with other biophysical techniques (patch-clamp, optical, and magnetic tweezers; atomic force microscopy; microfluidics) or photophysical effects are hence gaining attention. Although smFRET is particularly useful for the observation of dynamic conformational changes and subpopulations, FRET efficiencies also carry very precise information on the actual distance between fluorophores attached to distinct moieties of a macromolecule. As shown by recent work from many laboratories (such as those of Seidel, Michaelis, Hugel, and Grubmüller), this quantitative information can be used to help define biological structures and in the future should find a place in the protein database of molecular structures. smFRET has so far mostly been used for in vitro experiments but can be used additionally to monitor conformational dynamics and heterogeneity in live cells. “In vivo smFRET” has recently emerged as a promising methodology, demonstrated by the groups of Sakon, Weninger, Schuler, and Kapanidis among others. We envision that further technological developments will expand smFRET applications beyond dynamic structural biology to allow fast nonequilibrium kinetic studies, high-throughput drug screening, and molecular diagnostics. Advancements of these applications will be impactful for systems that are highly heterogeneous and dynamic.

Dynamic structural biology using smFRET.

Left: Principle of FRET as a molecular ruler. In a system with a pair of dyes, after the donor dye (D) is excited, it transfers the excitation energy to a nearby acceptor dye (A; top) with an efficiency (E) that depends on the sixth power of the distance between the dyes (bottom). Right: Use of FRET to study structural dynamics at the single-macromolecule level. The experimental setup (top), a combination of single-molecule fluorescence microscopy and spectroscopy, can be used to determine conformational states or dynamics in solution or on immobilized molecules. Here E is calculated per each single-molecule burst of photons, and bursts (n) are accumulated in E histograms (middle) or for different time bins to form a single-molecule E trajectory (bottom).


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.

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