Shcherbakova D.M. et al. J. Am. Chem. Soc. advance online publication (24 April 2012).

Imaging the interaction of multiple proteins tagged with different genetically encoded fluorescent proteins can require complex and expensive optical setups. One solution is to use fluorophores with a large Stokes shift between excitation and emission so that a single laser can be used to excite a number of markers with distinct emission wavelengths. Shcherbakova et al. filled a gap in the fluorophore spectrum by using random mutagenesis and directed evolution to engineer a bright monomeric orange fluorescent protein with a large Stokes shift, LSSmOrange (excitation/emission 437/572 nm). They demonstrated the utility of LSSmOrange by imaging four proteins simultaneously using fluorescence cross-correlation spectroscopy in vitro. They also designed two compatible pairs of Förster resonance energy transfer reporters to concurrently monitor calcium spikes and apoptosis in HeLa cells.