Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor.

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.

A long-wavelength fluorescent glucose biosensor based on bioconjugates of galactose/glucose binding protein and Nile Red derivatives.

BACKGROUND: Fluorescent biosensors based on galactose/glucose binding protein (GGBP) and environmentally sensitive derivatives of the phenoxazine dye Nile Red are described. These biosensors are proposed as the sensing platform for a minimally invasive, continuous glucose monitoring system that can be implanted under the skin and read transdermally using an external fluorometer. METHODS: To construct the biosensors, the thiol-reactive Nile Red derivatives INR and IANR were prepared and conjugated to GGBP proteins possessing cysteine mutations that were designed for optimal site-specific fluorophore attachment. The attachment sites were selected to maximize the local environment change for attached dyes between the bound and unbound conformations of GGBP. RESULTS: Fluorescence responses at the selected cysteine sites of GGBP upon binding to glucose showed that the conjugates typically yielded fluorescence emission around 640-650 nm with up to 50% changes in fluorescence intensity. Conjugate E149C/A213C/L238S INR GGBP also displayed glucose binding in the human physiological range (K (D) = 7.4 mM). CONCLUSIONS: The phenoxazine derivatives fluoresced at longer wavelengths (>600 nm) approaching the near-infrared spectral window, where interference from scattering and tissue absorbance are minimal. Ultimately, we expect that monitoring systems based on GGBP and longwavelength dyes will be implanted for up to 6 months and can be used to transmit information through the skin to an external monitor.

Fluorescence resonance energy transfer glucose sensor from site-specific dual labeling of glucose/galactose binding protein using ligand protection.

BACKGROUND: Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call "ligand protection." METHOD: In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents. The mutant E149C/A213C/L238S was first labeled at E149C in the presence of the ligand glucose. Following dialysis and removal of glucose, the protein was labeled with a second dye, either Texas Red (TR) C5 bromoacetamide or TR C2 maleimide, at the second site, A213C. RESULTS: Changes in glucose-dependent fluorescence were observed that were consistent with FRET between the nitrobenzoxadiazole and TR fluorophores. Comparison of models and spectroscopic properties of the C2 and C5 TR FRET constructs suggests the greater rigidity of the C2 linker provides more efficient FRET. CONCLUSIONS: The ligand protection strategy provides a simple method for labeling GGBP with two different fluorophores to construct FRET-based glucose sensors with glucose affinity within the human physiological glucose range (1-30 mM). This general strategy may also have broad utility for other protein-labeling applications.

Synthesis and biosensor performance of a near-IR thiol-reactive fluorophore based on benzothiazolium squaraine.

Environmentally sensitive near-IR (NIR) dyes are useful fluorophores for various biosensor applications when tissue absorption, scattering, and autofluorescence are a leading concern. Biosensors operating in the NIR region (generally wavelengths >650 nm) would avoid interference from biological media and thereby facilitate relatively interference free sensing. Squaraine dyes are potential candidates to serve as reporter molecules due to their spectral properties in the NIR region, but none is commercially available for site-specific coupling to proteins through native or engineered thiols on cysteine. In this context, we have synthesized a thiol-reactive squaraine that displays fluorescence emission above 650 nm and have coupled the dye site-specifically to various mutants of glucose/galactose binding protein that contained an engineered cysteine for attachment. Mutant E149C/A213R/L238S ISQ GGBP gave a fluorescence change of +50% and a binding constant of 12 mM, which is in the human physiological range for glucose.