Figure 25.

Overview of the properties and biophysical applications of flavin-binding fluorescent proteins (FbFPs). Constitutively fluorescent FbFPs are engineered from wild-type LOV domains, by substituting the active-site cysteine to abrogate canonical LOV photochemistry, and by introducing other mutations to increase the fluorescence quantum yield Φ_F. They can be used for imaging in fluorescence microscopy, as donors in FRET and as fluorescence-based sensors.^350–353 The photochromicity of cysteine-retaining LOV domains can be exploited in cellular super-resolution microscopy (nanoscopy),^157–159 while formation of the thioadduct in LOV domains underlies conventional optogenetic applications. In addition, FbFPs can function as genetically-encoded photosensitizers for ¹O₂, with a range of further applications.^354,355 Blue and purple arrows indicate excitation with blue or violet light, respectively.