Conformational Changes and Structure Formation of Proteins

Various single-molecule methods have been developed for the detection of different fluorescent observables. The oligomerization state of functional molecules and stoichiometry of different components within a complex can be quantified using the stepwise photobleaching of fluorescence trajectories (Ulbrich and Isacoff, 2007; Jiang et al., 2011). When this technique is combined with super-resolution microscopy, a single fluorophore can be localized with a precision of down to 10 nm, which can determine the spatial alignment of different subcomponents within one dense structure and the true size of the fine structures blurred by the diffraction limit (Hell and Wichmann, 1994; Betzig et al., 2006; Rust et al., 2006). To ascertain intramolecular conformational changes or intermolecular interactions, single-molecule Förster resonance energy transfer (smFRET) (Förster, 1948; Clegg, 1992) may be a suitable method. Energy transfer efficiency represents the distance between FRET donor and acceptor, resulting in a smFRET resolution of 3–10 nm, which is even less than the spatial resolution achieved using super-resolution microcopy (Ha et al., 1996).