Center for Plant Molecular Biology, Plant Physiology, University of Tübingen , Auf der Morgenstelle 32, 72076 Tübingen, Germany.
Institute for Physical and Theoretical Chemistry , Auf der Morgenstelle 18, 72076 Tübingen, Germany.
J Phys Chem B. 2017 Mar 23;121(11):2407-2419. doi: 10.1021/acs.jpcb.6b11623. Epub 2017 Mar 10.
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.
黄色荧光蛋白（YFP）常用于一种称为双分子荧光互补（BiFC）的蛋白质互补测定中，用于可视化蛋白质-蛋白质相互作用。在这种分析中，将 YFP 的两个不同的非荧光片段遗传连接到感兴趣的蛋白质上。当这些蛋白质相互作用时，YFP 片段被拉近到足够近的距离以重新形成其原始结构，从而产生荧光。BiFC 允许直接读取蛋白质-蛋白质相互作用，并且通过体内成像进一步促进其功能研究。此外，已经观察到，通过互补来自维多利亚水母绿色荧光蛋白的不同蛋白质的片段，可以扩展 BiFC 中的可用颜色范围，从而允许对蛋白质-蛋白质相互作用进行多重研究。以前已经报道了“多色”BiFC（mcBiFC）复合物的一些光谱特性；然而，尚未进行深入分析。因此，由于适当的表征主要依赖于体外数据，因此对这些 mcBiFC 复合物的光物理特性知之甚少。对于自荧光蛋白（AFP）的片段来说，这尤其困难，因为它们表现出非常强烈的形成超分子聚集体的趋势，这些聚集体在体外沉淀。在这项研究中，通过直接融合不同 AFP 片段的编码 DNA来克服这种内在困难。大肠杆菌中遗传序列的翻译导致具有独特性质的完全功能、高可溶性荧光蛋白。基于它们的构建，它们被指定为嵌合 AFP 或 BiFC 嵌合体。通过比较它们的光谱特性与实验体内 BiFC 数据，证实了嵌合蛋白作为 BiFC 模型系统的实用性。在这项研究中，在集合和单分子水平上对九种不同的嵌合体进行了彻底分析。数据表明，被认为在光物理上沉默的突变会显著改变 AFP 的性质。
