1 research outputs found
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 proteināprotein 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 proteināprotein 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 proteināprotein interactions.
Some spectral characteristics of āmulticolorā BiFC (mcBiFC)
complexes have been reported before; however, no in-depth analysis
has been performed yet. Therefore, little is known about the photophysical
characteristics of these mcBiFC complexes because a proper characterization
essentially relies on in vitro data. This is particularly difficult
for fragments of autofluorescent proteins (AFPs) because they show
a very strong tendency to form supramolecular aggregates which precipitate
ex vivo. In this study, this intrinsic difficulty is overcome by directly
fusing the coding DNA of different AFP fragments. Translation of the
genetic sequence in Escherichia coli leads to fully functional, highly soluble fluorescent proteins with
distinct properties. On the basis of their construction, they are
designated chimeric AFPs, or BiFC chimeras, here. Comparison of their
spectral characteristics with experimental in vivo BiFC data confirmed
the utility of the chimeric proteins as a BiFC model system. In this
study, nine different chimeras were thoroughly analyzed at both the
ensemble and the single-molecular level. The data indicates that mutations
believed to be photophysically silent significantly alter the properties
of AFPs