Head, Trajen - University of Miami Miller School of Medicine
Co-authors: Bachas, Leonidas - University of Miami, Cullinan, Megan - University of Miami Miller School of Medicine, Daunert, Sylvia - University of Miami Miller School of Medicine, Galvin, Connor - University of Miami Miller School of Medicine, Zahran, Elsayed M - University of Miami, Zeynaloo, Elnaz - University of Miami Miller School of Medicine
Glucose/Galactose Binding Protein (GBP) is a member of the periplasmic binding protein family, and is natively expressed by Escherichia coli. The structure of this binding protein consists of two globular domains connected by a short hinge region. Importantly, as its name implies, GBP is a naturally selective probe for glucose, and contains a binding site for this small molecule between its two globular domains. In the presence of glucose, these two domains close together around the glucose molecule, dramatically altering the conformation of the protein. Within this centrally located binding pocket are one of the five tryptophan (TRP) residues, and one of the seven phenylalanine (PHE) residues. We have previously shown that through the incorporation of non-natural amino acids (NNAA), it is possible to confer unique properties to the target protein. Specifically, we have previously developed glucose sensors possessing unique fluorescence characteristics through incorporation of the NNAAs 7-azatryptophan and 5-fluorotryptophan. In the current work, we demonstrate the successful global incorporation of 3,4-dihydroxyphenylalanin (L-DOPA) into GBP in the place of PHE. L-DOPA contains a catechol moiety that participates in a quasi-reversible, 2-electron redox process. The conformational change that occurs as a result of substrate binding buries the amino acid residues surrounding the binding pocket and alters their local environment. We demonstrate that this conformational change upon substrate binding is detectable as a change in the electrochemical behavior of incorporated L-DOPA residues, and that this newly synthesized electroactive GBP biosensor can be used for the selective electrochemical detection of glucose. To the best of our knowledge, this represents the first report of electrochemical detection of glucose via inherently electroactive amino acids incorporated into the primary sequence of a protein.
Application: Bioanalytical
Methodology: Electrochemistry