Construction of a fluorescent biosensor family

Abstract

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.

Fluorescence Resonance Energy Transfer Glucose Sensor from Site-Specific Dual Labeling of Glucose/Galactose Binding Protein Using Ligand Protection

“…The mechanism for the two dual-labeled NBD-TR CCS proteins appears to result primarily from the large change in fluorescence intensity upon glucose binding of the environment-sensitive donor fluorophore NBD at the E149C site 11 as well as a modest contribution from distancedependent FRET between NBD and TR. The absorbance spectra of NBD for both NBD-TR C2 CCS and NBD-TR C5 CCS (Figure 3) do not indicate that any ground state perturbation of NBD is occurring, unlike the case of doubly labeled 2NBD CCS, in which a significant blue shift is observed.…”

“…13,22 The potential of GGBP for in vivo blood glucose monitoring has been frequently suggested. 10,11,23,24 A prototype continuous glucose fiber optic sensor based on GGBP was evaluated in preclinical studies, 25 and the sensor has further demonstrated competitive accuracy in a human feasibility study. 26 The GGBP in the prototype sensor was labeled with a single environment-sensitive fluorophore, acrylodan.…”

Engineered Derivatives of Maltose-Binding Protein

“…They found a increase in fluorescence intensity of 1.8-fold with their sensor. Hellinga and coworkers studied this kind of biosensor in detail, both with MBP and with their derivative that binds zinc (de Lorimier et al, 2002, Hellinga & Marvin, 1998, Marvin & Hellinga, 2001). They mutated a number of MBP residues to cysteine, then tested a number of fluorescent dyes to find the best combination of position and dye.…”

Fluorescent Biosensors for Protein Interactions and Drug Discovery

“…The family of periplasmic binding proteins (PBPs) that presented a conformational change upon ligand binding has been used to create diverse biosensors based in the fluorescent incorporation by chemical modification with thiol-fluorescent reactive for cysteine. This work has been pioneered by Cass and col by introduction of fluorescent group into the maltoside binding protein (MBP) a PBP, and extensively developed by Hellinga and collaborators (de Lorimier, Smith et al 2002). This family of proteins presented a bilobated structure with high similarity, that present at least two conformers: an open form in absence of ligand and a closed state bound to its ligand (Fig.…”