Construction of a reagentless glucose biosensor using molecular exciton luminescence.
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 Escherichia coli glucose/galactose binding protein (GGBP) was used to engineer double-cysteine mutations that allowed selective covalent attachment of thiol-reactive dyes. Because GGBP undergoes a large conformational change on the addition of glucose, rational placement of these sites allowed glucose-dependent spatial realignment of the two fluorophores, which was monitored as a change in fluorescence intensity and extinction coefficients. Using targeted mutagenesis of the GGBP binding pocket, glucose biosensors were created to measure concentrations spanning five orders of magnitude (0.04-12,000 microM). The glucose biosensor retained its function in complex solutions that 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.
Der
Bryan S
BS
Department of Chemistry, Gottwald Science Center, University of Richmond, Richmond, VA 23173, USA.
Dattelbaum
Jonathan D
JD
D000042:Absorption; D001665:Binding Sites; D015374:Biosensing Techniques / Q000379:methods*; D001786:Blood Glucose; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D049449:Luminescence; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D012235:Rhodamines / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D017186:Titrimetry
D001786:Blood Glucose; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009004:Monosaccharide Transport Proteins; D050505:Mutant Proteins; D033902:Periplasmic Binding Proteins; D012235:Rhodamines; C023550:galactose-binding protein; C034996:tetramethylrhodamine iodoacetamide; D005947:Glucose; D003545:Cysteine