Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation

The yellow fluorescent protein (YFP) is frequently used in a protein complementation assay called bimolecular fluorescence complementation (BiFC), and is employed to visualize protein–protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein–protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein–protein interactions. Some spectral characteristics of “multicolor” BiFC (mcBiFC) complexes have been rep...