NER: Surface Assembly of Functional Protein Nanosensors

纳米结构分子传感器将在工业过程、医疗诊断和其他应用中报告高分辨率的空间和时间系统数据，而不会显著影响它们监控的系统。这些纳米传感器需要专门的元件来将传感器连接到目标上，组装结构框架，感知环境的特定物理或化学状态，并将这种状态转换为输出信号。蛋白质受体为纳米传感器的开发提供了一个范例，因为它们已经形成了执行这些功能的结构域。提出了一种基于细胞黏附复合体范式的蛋白质纳米传感器。在接收到适当的环境刺激后，传感器将自组装亚单位，以支持结构角色、感知信号和产生化学信息。该项目的初始目标是优化传感器亚单位与惰性固体基质的粘附性。谷胱甘肽-S-转移酶(GST)将被用来将GST融合蛋白粘连到谷胱甘肽上，谷胱甘肽是一种GST配体，共价连接到玻璃表面。下一个项目目标是展示在表面检测到GST融合蛋白的酶修饰。酪氨酸激酶配体的GST融合将附着在表面，并将使用放射性磷酸或专门识别磷酸化蛋白质的抗体来检测该配体的激活或磷酸化。该项目的最后阶段将调查传感器激活的检测，其基础是将结构和信号部件招募到激活的传感器亚单位。磷酸化的酪氨酸残基与SH2蛋白结构域结合。SH2结构域将与荧光蛋白融合，通过简单的荧光测量即可检测到传感器的激活。

Nanostructured molecular sensors will report high-resolution spatial and temporal system data during industrial processes, health care diagnostics, and other applications without significantly affecting the systems they monitor. These nanosensors require specialized elements for sensor attachment to the target, assembly of a structural framework, sensing a particular physical or chemical state of the environment, and transducing this state into an output signal. Protein receptors offer a paradigm for nanosensor development as they have developed domains that perform each of these functions. It is proposed to develop a protein-based nanosensor based on the paradigm of cell adhesion complexes. Upon receiving the appropriate environmental stimulus the sensor will self-assemble subunits for supporting structural roles, sensing signals, and generating chemical messages.The initial aim of the project is to optimize adhesion of a sensor subunit to an inert solid matrix. Glutathione-S-Transferase (GST) will be employed to adhere GST-fusion proteins to glutathione, a GST ligand, covalently linked to a glass surface. The next project goal is to demonstrate detection of an enzymatic modification of the GST fusion protein on the surface. A GST fusion of a tyrosine kinase ligand will be attached to the surface and activation, or phosphorylations, of the ligand will be detected using radioactive phosphate or antibodies that specifically recognize phosphorylated proteins. The final stage of the project will investigate detection of sensor activation based on recruitment of structural and signaling components to an activated sensor subunit. Phosphorylated tyrosine residues bind to SH2 protein domains. An SH2 domain will be fused to a fluorescent protein, enabling detection of sensor activation by a simple fluorescence measurement.