title: Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation.
abstract: 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 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.
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Peter
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Sven Zur
authors.initials: SZ
authors.lastname: Oven-Krockhaus
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Manikandan
authors.initials: M
authors.lastname: Veerabagu
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Virtudes Mira
authors.initials: VM
authors.lastname: Rodado
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Berendzen
authors.affiliation: Institute for Physical and Theoretical Chemistry , Auf der Morgenstelle 18, 72076 Tübingen, Germany.
authors.forename: Alfred J
authors.initials: AJ
authors.lastname: Meixner
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Harter
authors.affiliation: Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.|Berthold Technologies GmbH & Co. KG , Calmbacherstr. 22, 75323 Bad Wildbad, Germany.
authors.forename: Frank E
authors.identifier: 0000-0003-2138-3170
authors.initials: FE
authors.lastname: Schleifenbaum
mesh_terms: D017360:Arabidopsis; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D001419:Bacteria; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D008027:Light; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects*
chemical_list: C576644:ARR18 protein, Arabidopsis; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D050976:Basic-Leucine Zipper Transcription Factors; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C495668:bZIP63 protein, Arabidopsis; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins