Versatile scaffolds to visualize endogenous protein activation in living cells

多功能支架可可视化活细胞中的内源蛋白激活

• 批准号: 7899891
• 负责人: Klaus M. Hahn
• 金额: $ 28.04万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7899891
• 关键词: Address; Affinity; Bacteriophages; Binding; Biological; Biosensor; Cells; Characteristics; Computer Simulation; Coupled; Coupling; Cysteine; Dyes; Engineering; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Image; Immunoglobulin Variable Region; In Vitro; Interdisciplinary Study; Iodoacetamide; Label; Libraries; Life; MAPK1 gene; MAPK8 gene; Modeling; Molecular Models; Pathway interactions; Peptide Hydrolases; Phage Display; Phosphoproteins; Phosphorylation; Phosphotransferases; Positioning Attribute; Production; Protein Binding; Protein Binding Domain; Protein Engineering; Proteins; Reagent; Regulation; Reporting; Scaffolding Protein; Screening procedure; Sensitivity and Specificity; Series; Signal Transduction; Site; Site-Directed Mutagenesis; Structure; Surface; Technology; Testing; Validation; base; career; cell behavior; cellular imaging; design; directed evolution; experience; fluorophore; high throughput screening; improved; molecular modeling; mutant; novel; novel strategies; protein activation; quantum; response; scaffold; sensor; src-Family Kinases

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.

描述（由申请人提供）：该提案提出了一种新方法来产生荧光生物传感器，能够报告活细胞中内源性蛋白激活的时空动力学。生物传感器由“亲和力试剂”组成，该试剂专门与靶蛋白的活化形式结合，并与设计用于活细胞成像的新型染料结合。当亲和力试剂找到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。亲和力试剂的库将显示在噬菌体的表面上，从而为使用高吞吐量筛选的特定目标选择生物传感器。这种方法可以为以前无法​​访问的靶标生成生物传感器，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。该方法比其他当前的生物传感器设计都没有扰动，因为可以检查内源性蛋白质，并且因为明亮的染料直接激发了增强的敏感性。噬菌体显示可用于“微调”生物传感器的亲和力和可逆性。专注的文库将用于针对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，可变区域将被引入已经靶向磷蛋白的天然域，并将设计蛋白质支架作为生物传感器。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有效区域。我们将针对信号调节的两种广泛相关的机制：自身抑制性和激酶结构域的磷酸化和分子内相互作用。 SRC，PAK，JNK和ERK2的生物传感器将作为这种调节的示例，并且因为这些分子的生物传感器将使我们能够解决对这一新的生物传感器试剂的“现实世界”测试的重要生物学问题。 pak，jnk和erk2各自在SRC下游的不同平行路径上。将检查该信号网络的时空调节以产生不同的细胞反应。

项目成果

Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase.

高度特异性 FN3 单体定向进化为人 Lyn 酪氨酸激酶的 SH3 结构域。

• DOI: 10.1371/journal.pone.0145872
• 发表时间: 2016
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Huang,Renhua;Fang,Pete;Hao,Zengping;Kay,BrianK
• 通讯作者: Kay,BrianK

Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase.

• DOI: 10.1016/j.nbt.2011.11.015
• 发表时间: 2012-06-15
• 期刊: NEW BIOTECHNOLOGY
• 影响因子: 5.4
• 作者: Huang, Renhua;Fang, Pete;Kay, Brian K.
• 通讯作者: Kay, Brian K.