Award Abstract #0103348
NER: Surface Assembly of Functional Protein Nanosensors

NSF Org:	CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems
Initial Amendment Date:	July 20, 2001
Latest Amendment Date:	July 20, 2001
Award Number:	0103348
Award Instrument:	Standard Grant
Program Manager:	Fred G. Heineken CBET Division of Chemical, Bioengineering, Environmental, and Transport Systems ENG Directorate for Engineering
Start Date:	July 15, 2001
Expires:	December 31, 2002 (Estimated)
Awarded Amount to Date:	$71461
Investigator(s):	Sean Palecek palecek@engr.wisc.edu (Principal Investigator)
Sponsor:	University of Wisconsin-Madison 21 North Park Street MADISON, WI 53715 608/262-3822
NSF Program(s):	BIOCHEMICAL & BIOMASS ENG
Field Application(s):	0308000 Industrial Technology
Program Reference Code(s):	BIOT, 9181, 1676
Program Element Code(s):	1402

ABSTRACT

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.

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