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.

Bioconjugatable, PEGylated Hydroporphyrins for Photochemistry and Photomedicine. Narrow-Band, Near-Infrared-Emitting Bacteriochlorins.

Synthetic bacteriochlorins absorb in the near-infrared (NIR) region and are versatile analogues of natural bacteriochlorophylls. The utilization of these chromophores in energy sciences and photomedicine requires the ability to tailor their physicochemical properties, including the incorporation of units to impart water solubility. Herein, we report the synthesis, from two common bacteriochlorin building blocks, of five wavelength-tunable, bioconjugatable and water-soluble bacteriochlorins along with two non-bioconjugatable benchmarks. Each bacteriochlorin bears short polyethylene glycol (PEG) units as the water-solubilizing motif. The PEG groups are located at the 3,5-positions of aryl groups at the pyrrolic ß-positions to suppress aggregation in aqueous media. A handle containing a single carboxylic acid is incorporated to allow bioconjugation. The seven water-soluble bacteriochlorins in water display Qy absorption into the NIR range (679-819 nm), sharp emission (21-36 nm full-width-at-half-maximum) and modest fluorescence quantum yield (0.017-0.13). Each bacteriochlorin is neutral (non-ionic) yet soluble in organic (e.g., CH2Cl2, DMF) and aqueous solutions. Water solubility was assessed using absorption spectroscopy by changing the concentration ∼1000-fold (190-690 µM to 0.19-0.69 µM) with a reciprocal change in pathlength (0.1-10 cm). All bacteriochlorins showed excellent solubility in water, except for a bacteriochlorin-imide that gave slight aggregation at higher concentrations. One bacteriochlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The antibody conjugate of B2-NHS displayed a sharp signal upon ultraviolet laser excitation (355 nm) with NIR emission measured with a 730/45 nm bandpass filter. Overall, the study gives access to a set of water-soluble bacteriochlorins with desirable photophysical properties for use in multiple fields.

Bioconjugatable, PEGylated Hydroporphyrins for Photochemistry and Photomedicine. Narrow-Band, Red-Emitting Chlorins.

Chromophores that absorb and emit in the red spectral region (600-700 nm), are water soluble, and bear a bioconjugatable tether are relatively rare yet would fulfill many applications in photochemistry and photomedicine. Here, three molecular designs have been developed wherein stable synthetic chlorins - analogues of chlorophylls - have been tailored with PEG groups for use in aqueous solution. The designs differ with regard to order of the installation (pre/post-formation of the chlorin macrocycle) and position of the PEG groups. Six PEGylated synthetic chlorins (three free bases, three zinc chelates) have been prepared, of which four are equipped with a bioconjugatable (carboxylic acid) tether. The most effective design for aqueous solubilization entails facial encumbrance where PEG groups project above and below the plane of the hydrophobic disk-like chlorin macrocycle. The chlorins possess strong absorption at ~400 nm (B band) and in the red region (Qy band); regardless of wavelength of excitation, emission occurs in the red region. Excitation in the ~400 nm region thus provides an effective Stokes shift of >200 nm. The four bioconjugatable water-soluble chlorins exhibit Qy absorption/emission in water at 613/614, 636/638, 698/700 and 706/710 nm. The spectral properties are essentially unchanged in DMF and water for the facially encumbered chlorins, which also exhibit narrow Qy absorption and emission bands (full-width-at-half maximum of each <25 nm). The water-solubility was assessed by absorption spectroscopy over the concentration range ~0.4 µM - 0.4 mM. One chlorin was conjugated to a mouse polyclonal IgG antibody for use in flow cytometry with compensation beads for proof-of-principle. The conjugate displayed a sharp signal when excited by a violet laser (405 nm) with emission in the 620-660 nm range. Taken together, the designs described herein augur well for development of a set of spectrally distinct chlorins with relatively sharp bands in the red region.

Evaluation of the relative stability of liganded versus ligand-free protein conformations using Simplicial Neighborhood Analysis of Protein Packing (SNAPP) method.

Many proteins change their conformation upon ligand binding. For instance, bacterial periplasmic binding proteins (bPBPs), which transport nutrients into the cytoplasm, generally consist of two globular domains connected by strands, forming a hinge. During ligand binding, hinge motion changes the conformation from the open to the closed form. Both forms can be crystallized without a ligand, suggesting that the energy difference between them is small. We applied Simplicial Neighborhood Analysis of Protein Packing (SNAPP) as a method to evaluate the relative stability of open and closed forms in bPBPs. Using united residue representation of amino acids, SNAPP performs Delaunay tessellation of the protein, producing an aggregate of space-filling, irregular tetrahedra with nearest neighbor residues at the vertices. The SNAPP statistical scoring function is derived from log-likelihood scores for all possible quadruplet compositions of amino acids found in a representative subset of the Protein Data Bank, and the sum of the scores for a given protein provides the total SNAPP score. Results of scoring for bPBPs suggest that in most cases, the unliganded form is more stable than the liganded form, and this conclusion is corroborated by similar observations of other proteins undergoing conformation changes upon binding their ligands. The results of these studies suggest that the SNAPP method can be used to predict the relative stability of accessible protein conformations. Furthermore, the SNAPP method allows delineation of the role of individual residues in protein stabilization, thereby providing new testable hypotheses for rational site-directed mutagenesis in the context of protein engineering.

Direct detection of glucose by surface plasmon resonance with bacterial glucose/galactose-binding protein.

The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor. By site-specifically and covalently attaching a known receptor for glucose, the glucose/galactose-binding protein (GGBP), to the SPR surface, we were able to detect glucose binding and determine equilibrium binding constants. The site-specific coupling was accomplished by mutation of single amino acids on GGBP to cysteine and subsequent thiol conjugation. The resulting SPR surfaces had glucose-specific binding properties consistent with known properties of GGBP. Further modifications were introduced to weaken GGBP-binding affinity to more closely match physiologically relevant glucose concentrations (1-30 mM). One protein with a response close to this glucose range was identified, the GGBP triple mutant E149C, A213S, L238S with an equilibrium dissociation constant of 0.5mM. These results suggest that biosensors for direct glucose detection based on SPR or similar refractive detection methods, if miniaturized, have the potential for development as continuous glucose monitoring devices.

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.

Multi-day pre-clinical demonstration of glucose/galactose binding protein-based fiber optic sensor.

We report here the first pre-clinical demonstration of continuous glucose tracking by fluorophore-labeled and genetically engineered glucose/galactose binding protein (GGBP). Acrylodan-labeled GGBP was immobilized in a hydrogel matrix at the tip of a small diameter optical fiber contained in a stainless steel needle. The fiber optic biosensors were inserted subcutaneously into Yucatan and Yorkshire swine, and the sensor response to changing glucose levels was monitored at intervals over a 7-day period. Sensor mean percent error on day 7 was 16.4±5.0% using a single daily reference blood glucose value to calibrate the sensor. The GGBP sensor's susceptibility to common interferents was tested in a well-plate system using human sera. No significant interference was observed from the tested interferents except for tetracycline at the drug's maximum plasma concentration. The robust performance of the GGBP-based fiber optic sensor in swine models and resistance to interferents indicates the potential of this technology for continuous glucose monitoring in humans.

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.

Synthesis of thiol-reactive, long-wavelength fluorescent phenoxazine derivatives for biosensor applications.

Two environmentally sensitive, long-wavelength fluorescent phenoxazine derivatives, INR and IANR, were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins. The linkers were designed based on the attachment sites at two different positions on the phenoxazine, which were chosen in order to study the orientation of the dye with respect to the binding protein. Conjugation of the dyes to the S337C maltose binding protein (MBP) mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities. The dye INR gave a 3-fold (+200%) change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant (K(d)) of 435 microM. The fluorescence change for IANR was only 20% and the K(d) was 1.4 mM. Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye, resulting in little change in environment between the open and the closed-form conformations. The linker in INR, on the other hand, showed restricted motion, which placed the dye in different environments in the open and closed forms of the protein. Thus, design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.

Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration.

We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture.