Versatile scaffolds to visualize endogenous protein activation in living cells

描述（由申请人提供）：该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力学。这种生物传感器由一种“亲和试剂”组成，它能特异性地与靶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偶联。当亲和试剂发现并结合活化的靶标时，染料经历适合于比率成像的荧光变化。亲和试剂库将展示在噬菌体的表面上，使得能够使用高通量筛选来选择针对特定靶标的生物传感器。这种方法可以为以前无法接近的目标产生生物传感器，因为它不依赖于识别结合活化目标或已知目标底物的天然存在的蛋白质结构域。该方法比其他当前生物传感器设计更少干扰，因为可以检查内源性蛋白质，并且因为明亮的染料被直接激发以增强灵敏度。噬菌体展示可用于“微调”生物传感器的亲和力和可逆性。将产生聚焦文库以靶向特定类型的蛋白质靶标，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，可变区将被引入到已经靶向磷蛋白的天然存在的结构域中，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改善传感器的细胞内稳定性、标记和表达，并将可变性限制在结构的最高产区域。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ERK 2的生物传感器将作为这种调节的例子，并且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，从而对这类新的生物传感器试剂进行“真实的世界”测试。PAK、JNK和ERK 2各自在Src下游的不同的平行途径上。这个信号网络产生不同的细胞反应的时空调节将被检查。

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.