Student Research - Biochemistry

Bryan Der

Abstract: There is a continuing need for accurate and reliable glucose measurements in medical and industrial applications. Our research aims to design a new method to measure glucose concentrations in solution using a fluorescent protein biosensor. To develop this fluorescent biosensor, a protein that undergoes a conformational change after binding to glucose must be identified, the specific sites to attach a fluoroescent probe must be identified, and the fluorescence change after binding must be measured. The E. coli glucose binding protein (GBP) serves as the basis for our biosensor because of its high specificity for binding glucose and because GBP undergoes a significant conformational change after binding to glucose. Through molecular modeling and site-directed mutagenesis, we have constructed single and double cysteine mutants, which allow the covalent attachment of thiol-reactive fluorophores to opposite sides of the glucose binding pocket. Because of the large conformational change exhibited by GBP, the probes are close together before binding and then are pulled apart when GBP binds to glucose, resulting in increased fluorescence intensity. This data can then be converted into a standard curve that will enable us to accurately quantitate glucose concentrations. This developed technology may have applications in medicine, cell biology, environmental sciences, and bioprocess monitoring and may even one day replace the needle-stick method that diabetic patients use to monitor blood sugar levels.