Periplasmic binding proteins from bacteria such as Escherichia coli (E. coli) are important biomolecules which participate in the transport of substrates between the periplasm and the cytoplasm of the cells. In the presence of their respective substrates, the two globular domains of the proteins undergo a hinge motion completely engulfing the ligands. The short peptide links, which connect the two domains, act as the hinge during this structural rearrangement. These conformational changes that proteins such as the phosphate binding protein (PBP), sulfate binding protein (SBP), and galactose/glucose binding protein (GBP) undergo upon binding to their respective ligands can be used as the basis for the development of an optical sensing system for phosphate, sulfate, and glucose, respectively. The wild-type forms of these proteins lack cysteine residues in their primary structure. Using the polymerase chain reaction (PCR), single cysteines were introduced via site-directed mutagenesis. These mutant proteins were expressed in the periplasm and released by osmotic shock. Novel purification methods were developed whereby the proteins of interest were isolated from the crude extract in a single step using perfusion anion exchange chromatography. The unique cysteine residues were labeled with various environment-sensitive fluorophores such as N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), and N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester). Site-specific labeling ensures the reporting of conformational changes that the proteins undergo upon ligand binding. The changes in the fluorescence properties of the conjugates were monitored and related to the amount of ligand present. These steady-state fluorescence studies clearly indicated that the hinge motion and binding properties of these proteins could be utilized to develop a fluorescence-based biosensing system for phosphate, sulfate, and glucose.
ASJC Scopus subject areas
- Chemical Engineering(all)