Development of Fluorescent Protein Biosensors

描述（由申请人提供）：在短短十年的时间里，绿色荧光蛋白已经从完全默默无闻到在细胞生物学中作为蛋白质定位和基因表达的标记物占据主导地位。然而，使用工程GFP变体作为体内生化事件的实时指标可能对细胞生物学产生更大的影响。蛋白质工程和晶体学将用于开发和改进荧光蛋白指示器，作为活细胞中氧化还原事件和pH值的可视化报告。现有的氧化还原生物传感器将被重新设计，其目标是：(1)扩大其覆盖范围，以获得氧化亚细胞区室的氧化还原电位；(2)提高其对作为瞬时第二信使的活性氧（ROS）的敏感性。一类新的嵌合fret基红绿氧化还原指示剂将被开发用于细胞分选应用。合作者已经同意在酵母、植物和哺乳动物细胞中测试新的结构。

DESCRIPTION (provided by applicant): In just one decade, green fluorescent protein has rocketed from complete obscurity to dominate the scene in cell biology as a marker for protein localization and gene expression. However, the use of engineered GFP variants as real-time indicators of biochemical events in vivo could have even greater impact on cell biology. Protein engineering and crystallography will be used to develop and improve fluorescent protein indicators for use as visual reporters of redox events and pH in living cells. Available redox biosensors will be redesigned with goals (1) to extend their range of coverage to the redox potentials that obtain in oxidizing subcellular compartments and (2) to increase their sensitivity toward reactive oxygen species (ROS) that act as transient second messengers. A new class of chimeric FRET-based red-green redox indicator will be developed for use in cell-sorting applications. Collaborators have agreed to test new constructions in yeast, plants and mammalian cells. The mechanisms of redox-sensitive GFPs will be investigated. Fluorescent proteins are suspected to generate low levels of ROS that are toxic to cells under certain conditions. The number and nature of probe-generated ROS will be identified and their effects included in analysis of probe response. Finally, the chromophore of GFP is an example of a hydroxyarene photoacid, compounds that can exhibit excited state acidities similar to mineral acids while having a pKa near neutrality in the ground state. GFP provides a unique biological system for study of excited state proton transfer (ESPT) which, in turn, is central to the mechanism of GFP indicators. In a collaborative effort, atomic models and mechanisms for ESPT pathways will be tested for several examples of indicator GFPs using directed mutagenesis, crystallography and ultrafast time-resolved fluorescence spectroscopy.