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Date of Award

2008

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biochemistry & Molecular Biol.

Abstract

Fluorescent protein biosensors, which exhibit a significant change in fluorescence based on the physical interaction between protein and ligand, may prove to be effective tools to measure a variety of analytes. In particular, real-time monitoring of glucose levels has potential applications in bioprocess monitoring and in minimizing health complications caused by diabetes. In this work, site-directed mutagenesis of the E. coli glucose/galactose binding protein (GGBP) was used to engineer double cysteine mutations that allowed selective covalent attachment of thiol-reactive dyes. Since GGBP undergoes a large conformational change upon the addition of glucose, rational placement of these sites allowed for glucose-dependent spatial realignment of the two fluorophores for H-type dimer formation, which was monitored as a change in fluorescence intensity and extinction coefficients. Using targeted mutagenesis of the GGBP binding pocket, these sensors can be used to measure glucose concentrations spanning five orders of magnitude (0.04 to 12,000 μM). The glucose biosensor retained its function in complex solutions which contained realistic concentrations of protein and potential interfering agents found in blood serum. In addition to the development of a fluorescent protein sensor for glucose, this work helps to expand the spectroscopic tools used for the detection of conformational movements within a single polypeptide chain.

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