Essential cellular processes such as interactions between biomolecules or conformational changes often involve distance changes at a nanometer length scale (1–10 nm). Fluorescence resonance energy transfer (FRET) (Forster, 1949; Stryer & Haugland, 1967) is a spectroscopic technique that enables the observation of distance changes at this length scale with high sensitivity and in real time. In this technique, a donor and an acceptor fluorophore are introduced at sites whose distance is to be monitored. Upon excitation of the donor fluorophore, a fraction of its energy can be transferred to the acceptor fluorophore in a nonradiative process. The efficiency of energy transfer, E, is highly sensitive to the distance R between the two fluorescent molecules. FRET measurements at the single-molecule level allow the observation of dynamics on a molecular scale that would be inaccessible in ensemble measurements due to random averaging (Ha, 2001; Ha et al., 1996; Zhuang et al., 2000). The ability to monitor conformational dynamics of individual molecules in real time makes single-molecule techniques highly effective in the study of a wide range of mechanistic questions.
Adapted from: Deindl & Zhuang, 2012
We study the structural architecture of molecular machines using various techniques including X-ray crystallography, cryo-electron microscopy, small angle X-ray scattering (SAXS), and cross-linking mass spectrometry.
Ultimately, we hope to combine structural data and biochemistry with real-time dynamic information from single-molecule experiments in order to provide a more complete quantitative and mechanistic understanding of molecular machines.