NER: Surface Assembly of Functional Protein Nanosensors

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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.