A novel reagentless sensing system for measuring glucose based on the galactose/glucose-binding protein.

The galactose/glucose-binding protein (GBP) is synthesized in the cytoplasm of Escherichia coli in a precursor form and exported into the periplasmic space upon cleavage of a 23-amino-acid leader sequence. GBP binds galactose and glucose in a highly specific manner. The ligand induces a hinge motion in GBP and the resultant protein conformational change constitutes the basis of the sensing system. The mglB gene, which codes for GBP, was isolated from the chromosome of E. coli using the polymerase chain reaction (PCR). Since wild-type GBP lacks cysteines in its structure, introducing this amino acid by site-directed mutagenesis ensures single-label attachment at specific sites with a sulfhydro-specific fluorescent probe. Site-directed mutagenesis by overlap extension PCR was performed to prepare three different mutants to introduce a single cysteine residue at positions 148, 152, and 182. Since these residues are not involved in ligand binding and since they are located at the edge of the binding cleft, they experience a significant change in environment upon binding of galactose or glucose. The sensing system strategy is based on the fluorescence changes of the probe as the protein undergoes a structural change on binding. In this work a reagentless sensing system has been rationally designed that can detect submicromolar concentrations of glucose. The calibration plots have a linear working range of three orders of magnitude. Although the system can sense galactose as well, this epimer is not a potential interfering substance since its concentration in blood is negligible.

A dynamical investigation of acrylodan-labeled mutant phosphate binding protein.

The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigated through steady-state and time-resolved fluorescence spectroscopy. As a means of developing a biorecognition element for inorganic phosphate (P(i)), alanine-197 of PBP was replaced with a cysteine. This site was then labeled with an environmentally sensitive fluorophore. The fluorescence emission of the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P(i), as indicated by a 50% increase in the steady-state emission intensity. Steady-state results indicated that the labeling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed. Time-resolved measurements were used to determine the influence of the dynamics of MPBP-AC on the process of signal transduction. Time-resolved anisotropy measurements revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local motion of the acrylodan probe. The rates of both global and local rotational reorientation of MPBP-AC were faster when the protein was P(i)-bound rather than P(i)-free. This was a result of structural changes involving or surrounding both the P(i)-binding site (global changes) and the residues in near proximity to the fluorescent reporter group (local changes). Recovery of the semiangle (theta) indicated that local structural changes in MPBP-AC took place when P(i) was bound to the protein. Acrylodan gained mobility when MPBP-AC bound P(i), as indicated by the fact that theta increased by approximately 5 degrees. In addition, dynamic quenching measurements confirmed that structural changes occurred locally near the cys-197. Acrylodan became more accessible to iodide when MPBP-AC bound P(i), as demonstrated by the 35% increase in the value of the bimolecular quenching constant.