RESUMO
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.
Assuntos
Proteínas de Arabidopsis/efeitos da radiação , Fatores de Transcrição de Zíper de Leucina Básica/efeitos da radiação , Proteínas Luminescentes/efeitos da radiação , Fragmentos de Peptídeos/efeitos da radiação , Proteínas Recombinantes de Fusão/efeitos da radiação , Fatores de Transcrição/efeitos da radiação , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Bactérias , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/efeitos da radiação , Concentração de Íons de Hidrogênio , Luz , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
During photosynthesis, energy conversion at the two photosystems is controlled by highly complex and dynamic adaptation processes triggered by external factors such as light quality, intensity, and duration, or internal cues such as carbon availability. These dynamics have remained largely concealed so far, because current analytical techniques are based on the investigation of isolated chloroplasts lacking full adaptation ability and are performed at non-physiologically low temperatures. Here, we use non-invasive in planta spectro-microscopic approaches to investigate living chloroplasts in their native environment at ambient temperatures. This is a valuable approach to study the complex function of these systems, because an intrinsic property-the fluorescence emission-is exploited and no additional external perturbations are introduced. Our analysis demonstrates a dynamic adjustment of not only the photosystemI/photosystemII (PSI/PSII) intensity ratio in the chloroplasts but also of the capacity of the LHCs for energy transfer in response to environmental and internal cues.
