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Bimolecular Fluorescence Complementation
Published on: April 15, 2011
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Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence
Sébastien Peter1, Sven Zur Oven-Krockhaus1, Manikandan Veerabagu1
1Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
The Journal of Physical Chemistry. B
|February 28, 2017
Summary
This study introduces chimeric autofluorescent proteins (AFPs) for advanced bimolecular fluorescence complementation (BiFC) assays. These novel BiFC chimeras overcome aggregation issues, enabling detailed photophysical analysis and improved visualization of protein interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Bimolecular fluorescence complementation (BiFC) uses yellow fluorescent protein (YFP) fragments to visualize protein interactions in vivo.
- Extending the color range of BiFC (mcBiFC) allows multiplexed interaction studies, but photophysical characterization is challenging due to protein aggregation.
- Autofluorescent proteins (AFPs) are prone to aggregation, hindering in vitro analysis of mcBiFC complexes.
Purpose of the Study:
- To overcome challenges in analyzing multicolor BiFC (mcBiFC) complexes.
- To characterize the photophysical properties of novel chimeric AFPs (BiFC chimeras) as a model system for mcBiFC.
- To investigate the impact of mutations on AFP properties within the BiFC context.
Main Methods:
- Genetically fused coding DNA of different AFP fragments in Escherichia coli to create soluble, functional chimeric AFPs (BiFC chimeras).
- Thorough ensemble and single-molecular level analysis of nine different BiFC chimeras.
- Comparison of chimeric protein spectral characteristics with in vivo BiFC data.
Main Results:
- BiFC chimeras are highly soluble and functional, serving as a reliable model for mcBiFC.
- Detailed photophysical characterization of nine BiFC chimeras was achieved, overcoming previous limitations.
- Mutations previously considered photophysically silent were found to significantly alter AFP properties.
Conclusions:
- Chimeric AFPs provide a robust system for studying mcBiFC complexes.
- The study provides critical in vitro data for understanding mcBiFC photophysics.
- Even subtle mutations can have significant impacts on AFP spectral characteristics, relevant for BiFC assay design.
