Versatile scaffolds to visualize endogenous protein activation in living cells

描述（由申请人提供）：本提案提出了一种生产荧光生物传感器的新方法，能够报告活细胞中内源性蛋白质激活的时空动态。这种生物传感器由一种“亲和试剂”组成，它可以特异性地与目标蛋白的活化形式结合，并与一种专为活细胞成像而设计的新型染料结合。当亲和试剂发现并结合活化的靶时，染料发生适合比率成像的荧光变化。亲和试剂库将显示在噬菌体表面，使生物传感器选择特定的目标使用高通量筛选。这种方法可以为以前无法接近的靶标生产生物传感器，因为它不依赖于识别结合活化靶标或已知靶标底物的天然存在的蛋白质结构域。这种方法比目前其他生物传感器设计的干扰更小，因为内源性蛋白质可以被检测，而且明亮的染料被直接激发以提高灵敏度。噬菌体展示可用于“微调”生物传感器的亲和力和可逆性。针对特定类型的蛋白质靶点，将产生有针对性的文库，从而提高筛选效率和生物传感器的结合特性。通过计算和蛋白质建模，可变区域将被引入到已经针对磷蛋白的自然发生的区域中，蛋白质支架将被设计用作生物传感器。计算和蛋白质建模将用于提高传感器的细胞内稳定性，标记和表达，并将变异性限制在结构的最多产区域。我们将针对两个广泛相关的信号调节机制：磷酸化和自抑制和激酶结构域的分子内相互作用。Src， PAK， JNK和ERK2的生物传感器将作为这种调节的例子，因为这些分子的生物传感器将使我们能够解决一个重要的生物学问题，对于这种新型生物传感器试剂的“现实世界”测试。PAK、JNK和ERK2分别位于Src下游不同的平行通路上。这个信号网络的时空调节产生不同的细胞反应将被检查。

DESCRIPTION (provided by applicant): This proposal presents a new approach to produce fluorescent biosensors, capable of reporting the spatio-temporal dynamics of endogenous protein activation in living cells. The biosensors consist of an 'affinity reagent' which binds specifically to the activated form of the target protein, coupled to a novel dye designed for live cell imaging. The dye undergoes a fluorescence change suitable for ratio imaging when the affinity reagent finds and binds the activated target. Libraries of affinity reagents will be displayed on the surface of phage, enabling selection of biosensors for specific targets using high throughput screening. This approach can produce biosensors for previously inaccessible targets, because it does not rely on identifying naturally occurring protein domains that bind activated target, or known target substrates. The approach is less perturbing than other current biosensor designs both because endogenous proteins can be examined, and because the bright dyes are directly excited for enhanced sensitivity. Phage display can be used to 'fine tune' the affinity and reversibility of the biosensor. Focused libraries will be produced to target a particular type of protein target, thus improving the efficiency of screening and the binding characteristics of the biosensor. Through computation and protein modeling, variable regions will be introduced into naturally occurring domains already targeted to phosphoproteins, and protein scaffolds will be engineered for use as biosensors. Computation and protein modeling will be used to improve the intracellular stability, labeling and expression of the sensors, and to restrict variability to the most productive regions of the structure. We will target two broadly relevant mechanisms of signaling regulation: phosphorylation and intramolecular interaction of autoinhibitory and kinase domains. Biosensors for Src, PAK, JNK, and ERK2 will be targeted as examples of such regulation, and because biosensors of these molecules will enable us to address an important biological question, for a 'real world' test of this new class of biosensor reagents. PAK, JNK, and ERK2 are each on a different, parallel pathway downstream of Src. The spatio-temporal regulation of this signaling network to produce different cellular responses will be examined.