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.
Bibliographical noteFunding Information:
We acknowledge contributions and discussions with past and present members of the Weiss lab. We thank M. Segal for editing the manuscript. Supported by NIH grants GM069709 and NSF grant MCB-1244175 (S.W.), NIH grant GM095904 (X.M. and S.W.), and European Research Council grant ERC-STG 638536–SM-IMPORT, grant of the Deutsche Forschungsgemeinschaft within GRK2062, the Center for NanoScience, Center for Integrated Protein Science Munich, and LMUexcellent (T.C.).