Development of Fluorescent Protein Biosensors

描述（由申请人提供）：在短短十年内，绿色荧光蛋白作为蛋白定位和基因表达的标记物从完全默默无闻迅速发展到在细胞生物学中占据主导地位。然而，使用工程化的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剂，用作活细胞中氧化还原事件和pH值的视觉报告者。现有的氧化还原生物传感器将被重新设计，目标是（1）将其覆盖范围扩展到氧化亚细胞区室中获得的氧化还原电位，以及（2）增加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为基础的红-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。 将研究氧化还原敏感性GFP的机制。荧光蛋白被怀疑在某些条件下产生对细胞有毒的低水平的ROS。将确定探针产生的ROS的数量和性质，并将其影响纳入探针响应分析中。最后，GFP的发色团是羟基芳烃光酸的一个例子，这种化合物可以表现出类似于无机酸的激发态酸性，同时在基态具有接近中性的pKa。GFP为研究激发态质子转移（ESPT）提供了一个独特的生物系统，而ESPT又是GFP指示剂机制的核心。在一项合作努力中，ESPT途径的原子模型和机制将使用定向诱变，晶体学和超快时间分辨荧光光谱法测试指示GFP的几个例子。

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.