Glucose sensing is a critical health care need. The primary sensing moiety that has been used for monitoring blood glucose has been the enzyme glucose oxidase. Unfortunately, this technique requires a sample of fresh blood to be drawn. Ultimately, sensing strategies that can be interfaced to closed-loop insulin delivery systems will require an indwelling sensor. The glucose binding protein Concanavalin A has been extensively studied for its potential use in such a system, but has thus far failed to work. Clearly, a different approach is needed. Here we propose to take advantage of the ability of the glucose-galactose binding protein (GGBP) from E. coli to bind glucose and undergo dramatic conformational changes upon binding. The most common technique for transforming a protein into a fluorescent biosensor is by randomly labeling it with fluorescent dyes. The disadvantages of this random technique are numerous, but the most important is that the label may be placed at a site that is not sensitive to the environmental changes that we wish to detect. The novelty of our project is that we will site- specifically label GGBP, by site-directed mutagenesis at just the right positions for maximum response to glucose levels in the environment with minimum perturbation of protein activity. A fluorescent donor and an absorbing acceptor dye will be attached to cysteine mutations at positions 182 and 26 of the protein. These amino acids are located at opposite domains of the GGBP molecule. In the absence of glucose, GGBP is free to twist about its axis thereby allowing the donor and acceptor dyes to get close enough for fluorescence resonance energy transfer (FRET). Upon glucose binding, GGBP assumes a more compact and more rigid structure, with the donor and acceptor dyes facing opposite sides of the molecule. The result is a decrease in FRET with increasing concentrations of glucose. The degree of FRET is correlated to the average decay time of the donor fluorophore measured by the phase- modulation method. We have assembled a team skilled in molecular biology, fermentation and scaleup, spectroscopy and protein engineering to tackle this problem. We believe that a superior reagentless glucose sensor can be made and can have a significant impact on the ability to move towards an indwelling glucose monitoring system.
| Gryczynski, I; Gryczynski, Z; Rao, G et al. (1999) Polarization-based oxygen sensor. Analyst 124:1041-4 |
