Douglas B. Sherman

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.

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 μM 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.

Real-time pathogen monitoring during enrichment: a novel nanotechnology-based approach to food safety testing

We describe a new approach for the real-time detection and identification of pathogens in food and environmental samples undergoing culture. Surface Enhanced Raman Scattering (SERS) nanoparticles are combined with a novel homogeneous immunoassay to allow sensitive detection of pathogens in complex samples such as stomached food without the need for wash steps or extensive sample preparation. SERS-labeled immunoassay reagents are present in the cultural enrichment vessel, and the signal is monitored real-time through the wall of the vessel while culture is ongoing. This continuous monitoring of pathogen load throughout the enrichment process enables rapid, hands-free detection of food pathogens. Furthermore, the integration of the food pathogen immunoassay directly into the enrichment vessel enables fully biocontained food safety testing, thereby significantly reducing the risk of contaminating the surrounding environment with enriched pathogens. Here, we present experimental results showing the detection of E. coli, Salmonella, or Listeria in several matrices (raw ground beef, raw ground poultry, chocolate milk, tuna salad, spinach, brie cheese, hot dogs, deli turkey, orange juice, cola, and swabs and sponges used to sample a stainless steel surface) using the SERS system and demonstrate the accuracy of the approach compared to plating results.

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. Proteins 2004.

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.

Design and synthesis of a squaraine dye for long wavelength fluorescence-based biosensors

The design and synthesis of an environmentally sensitive long wavelength fluorescing squaraine dye for conjugation to proteins is dsecribed. Environmentally sensitive dyes are valuable for probing environmental changes that occur when labeled proteins bind their corresponding ligands and can be used to construct flyorescent sensors. Long wavelength (>650 nm) dyes would enable through-skin wireless sensing with minimum interference from the background. While several environmentally sensitive dyes are known in the visible spectrum, only a few are available in the long wavelength region, and none of them are available with reactive groups suitable for protein conjugation. Several derivatives of squarain dyes are known to be environmentally sensitive and fluorescent in the long wavelength region, but none of them are available with linkers for protein conjugation. In order to achieve this goal, we developed a synthetic scheme to introduce a reactive linker onto an anilinic squaraine that is highly sensitive to its environment. The synthesis involves the preparation of the dye with an iodoacetyl ester linker that readily reacts with a thiol on a cysteine residue of the binding protein. The squaraine dye was conjugated to known binding proteins that were evaluated as optical sensors. Ultimately, we expect these systems to measure analytes in the body and transmit information through the skin to an external monitor.