NER: Synthesis and Characterization of Genetically Engineered Protein Polymers: Effects of Sequence Disorder on Structure-property Relationships

NER：基因工程蛋白质聚合物的合成和表征：序列无序对结构-性质关系的影响

• 批准号: 0103468
• 负责人: Susan Muller
• 金额: $ 9.98万
• 依托单位: University of California-Berkeley
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-15 至 2003-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0103468&HistoricalAwards=false
• 关键词: NER; Synthesis; Characterization; Genetically; Engineered

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.

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所监测的系统。 这些纳米传感器需要专门的元件，用于将传感器附着到目标，组装结构框架，感测环境的特定物理或化学状态，并将此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，因为它们已经开发了执行这些功能的域。 提出了基于细胞粘附复合物的范例开发基于蛋白质的纳米传感器。 在接收到适当的环境刺激后，传感器将自组装亚基，以支持结构角色，传感信号，并产生化学信息。该项目的最初目标是优化传感器亚基与惰性固体基质的粘附。 谷胱甘肽-S-转移酶（GST）将用于将GST融合蛋白粘附于谷胱甘肽（一种共价连接至玻璃表面的GST配体）。 下一个项目目标是证明检测表面上GST融合蛋白的酶修饰。 将酪氨酸激酶配体的GST融合物连接到表面，并使用特异性识别磷酸化蛋白质的放射性磷酸盐或抗体检测配体的活化或磷酸化。 该项目的最后阶段将研究传感器激活的基础上招聘的结构和信号组件激活传感器亚基的检测。 磷酸化的酪氨酸残基与SH 2蛋白结构域结合。 SH 2结构域将与荧光蛋白融合，从而能够通过简单的荧光测量来检测传感器激活。