Detection of glucose-induced conformational change in hexokinase II using fluorescence complementation assay

Conformational changes in hexokinase are induced by its binding to glucose, thus providing an excellent example of an ‘induced fit’ model. To observe glucose-induced fluorescence restoration in hexokinase II using split-enhanced, green fluorescent protein (EGFP) in a process involving the reconstitution of split EGFP, E. coli cells expressing the chimeric NEGFP:HXK:CEGFP recombinant protein were treated with glucose and visualized via fluorescence read-outs. The reconstituted EGFP generated a strong fluorescence upon glucose stimulation of the bacteria. Moreover, the fluorescence intensity became stronger with increasing glucose up to 10 mM, with a maximum being observed after 60 min in a time- and concentration-dependent manner. Conformational changes associated with glucose-induced fit in human hexokinase II can thus be monitored successfully in vivo via fluorescence reconstitution assays, coupled with a quick and easy fluorescent read-out protocol.     

Fluorescence-based sensing of glucose using engineered glucose/galactose-binding protein: a comparison of fluorescence resonance energy transfer and environmentally sensitive dye labelling strategies

Fluorescence-based glucose sensors using glucose-binding protein (GBP) as the receptor have employed fluorescence resonance energy transfer (FRET) and environmentally sensitive dyes, but with widely varying sensitivity. We therefore compared signal changes in (a) a FRET system constructed by transglutaminase-mediated N-terminal attachment of Alexa Fluor 488/555 as donor and QSY 7 as acceptor at Cys 152 or 182 mutations with (b) GBP labelled with the environmentally sensitive dye badan at C152 or 182. Both FRET systems had a small maximal fluorescence change at saturating glucose (7% and 16%), badan attached at C152 was associated with a 300% maximal fluorescence increase with glucose, though with badan at C182 there was no change. We conclude that glucose sensing based on GBP and FRET does not produce a larger enough signal change for clinical use; both the nature of the environmentally sensitive dye and its site of conjugation seem important for maximum signal change; badan-GBP152C has a large glucose-induced fluorescence change, suitable for development as a glucose sensor.     

Glucose sensing based on the intrinsic fluorescence of sol-gel immobilized yeast hexokinase

In this study, we investigated measurements of the intrinsic fluorescence of yeast hexokinase as an assay for glucose and immobilization of the enzyme in a silica sol-gel matrix as a potential in vivo glucose sensor for use in patients with diabetes. The intrinsic fluorescence of hexokinase in solution (excitation=295 nm, emission=330 nm) decreased by 23% at a saturating glucose concentration of 1 mM (Kd=0.3 mM), but serum abolished the glucose-related fluorescence response. When entrapped in tetramethylorthosilicate-derived sol gel, hexokinase retained activity, with a 25% maximal glucose-related decrease in intrinsic fluorescence, and the saturation point was increased to 50 mM glucose (Kd=12.5 mM). The glucose response range was increased further (to 120 mM, Kd=57 mM) by a covering membrane of poly(2-hydroxyethyl) methacrylate. Unlike free enzyme, the fluorescence responses to glucose with sol-gel immobilized hexokinase, with or without covering membrane, were similar for buffer and serum. We conclude that fluorescence monitoring of sol-gel entrapped yeast hexokinase is a suitable system for development as an in vivo glucose biosensor.     

Construction of a reagentless glucose biosensor using molecular exciton luminescence

Fluorescent protein biosensors, which exhibit a significant change in fluorescence based on the physical interaction between protein and ligand, may prove to be effective tools to measure a variety of analytes. In particular, real-time monitoring of glucose levels has potential applications in bioprocess monitoring and in minimizing health complications caused by diabetes. In this work, site-directed mutagenesis of the Escherichia coli glucose/galactose binding protein (GGBP) was used to engineer double-cysteine mutations that allowed selective covalent attachment of thiol-reactive dyes. Because GGBP undergoes a large conformational change on the addition of glucose, rational placement of these sites allowed glucose-dependent spatial realignment of the two fluorophores, which was monitored as a change in fluorescence intensity and extinction coefficients. Using targeted mutagenesis of the GGBP binding pocket, glucose biosensors were created to measure concentrations spanning five orders of magnitude (0.04-12,000 microM). The glucose biosensor retained its function in complex solutions that contained realistic concentrations of protein and potential interfering agents found in blood serum. In addition to the development of a fluorescent protein sensor for glucose, this work helps to expand the spectroscopic tools used for the detection of conformational movements within a single polypeptide chain.     

In vivo glucose monitoring: the clinical reality and the promise

Glucose monitoring is an essential component of modern diabetes management. Three in vivo glucose sensors are now available for clinical use: a subcutaneously implanted amperometric enzyme electrode, a reverse iontophoresis system and a microdialysis-based device. Improvements in glucose-sensing technology continue to be sought, e.g. wired enzyme technology, viscometric affinity sensing and totally implanted glucose sensors. Non-invasive glucose sensing is the ultimate goal of glucose monitoring, but the most investigated approach, near-infrared (NIR) spectroscopy, is presently too imprecise for clinical application. Fluorescence-based glucose sensing offers several advantages and we are investigating strategies which include NIR-based fluorescence resonance energy transfer using concanavalin A/dextran; changes in the intrinsic fluorescence of hexokinase encapsulated in sol-gel; and non-invasive glucose monitoring of cells by measuring glucose-related changes in NADP(H).     

Fluorescence intensity- and lifetime-based glucose sensing using an engineered high-Kd mutant of glucose/galactose-binding protein

We synthesized mutants of glucose/galactose-binding protein (GBP), labeled with the environmentally sensitive fluorophore Badan, with the aim of producing a fluorescence-based glucose sensing system with an operating range compatible with continuous glucose monitoring in patients with diabetes mellitus. From five mutants tested, the triple mutant H152C/A213R/L238S-Badan showed a large (200%) maximal increase in fluorescence intensity on the addition of glucose, with a binding constant (K_d) of 11 mM, an operating range of approximately 1-100 mM, and similar responses in buffer and serum. The mean fluorescence lifetime of this mutant also increased by 70% on the addition of glucose. We conclude that the GBP mutant H152C/A213R/L238S, when labeled with Badan, is suitable for development as a robust sensor for in vivo glucose monitoring in diabetes.     

One-Line Monitoring of Extracellular Brain Glucose Using Microdialysis and a NADPH-Linked Enzymatic Assay

A method to monitor extracellular glucose in freely moving rats, based on intracerebral microdialysis coupled to an enzyme reactor is described. The dialysate is continuously mixed with a solution containing the enzymes hexokinase and glucose-6-phosphate dehydrogenase, and the fluorescence of NADPH formed enables the on-line registration of extracellular glucose. The method is applied to monitor changes in extracellular brain glucose during the infusion of glucose, electrically induced seizure, immobilization stress, and repetitive hypoxia. After glucose loading or after seizure, hippocampus dialysate glucose concentration was increased transiently. During immobilization, there was a short-lasting decrease and, thereafter, an increase in the extracellular hippocampus glucose. During repetitive hypoxia in rats with a unilaterally occluded carotid artery, the content of glucose of striatal dialysates followed closely changes in blood pressure. These results illustrate the usefulness of the method in studying changes in brain glucose concentrations under pathological and physiological conditions.     

Fluorescence as a method to reveal structures and membrane-interactions of amyloidogenic proteins

Amyloidogenesis is a characteristic feature of the 40 or so known protein deposition diseases, and accumulating evidence strongly suggests that self-association of misfolded proteins into either fibrils, protofibrils, or soluble oligomeric species is cytotoxic. The most likely mechanism for toxicity is through perturbation of membrane structure, leading to increased membrane permeability and eventual cell death. There have been a rather limited number of investigations of the interactions of amyloidogenic polypeptides and their aggregated states with membranes; these are briefly reviewed here. Amyloidogenic proteins discussed include A-beta from Alzheimer's disease, the prion protein, α-synuclein from Parkinson's disease, transthyretin (FAP, SSA amyloidosis), immunoglobulin light chains (primary (AL) amyloidosis), serum amyloid A (secondary (AA) amyloidosis), amylin or IAPP (Type 2 diabetes) and apolipoproteins. This review highlights the significant role played by fluorescence techniques in unraveling the nature of amyloid fibrils and their interactions and effects on membranes. Fluorescence spectroscopy is a valuable and versatile method for studying the complex mechanisms of protein aggregation, amyloid fibril formation and the interactions of amyloidogenic proteins with membranes. Commonly used fluorescent techniques include intrinsic and extrinsic fluorophores, fluorescent probes incorporated in the membrane, steady-state and lifetime measurements of fluorescence emission, fluorescence correlation spectroscopy, fluorescence anisotropy and polarization, fluorescence resonance energy transfer (FRET), fluorescence quenching, and fluorescence microscopy.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.     

Glucose biosensors as models for the development of advanced protein-based biosensors

Glucose sensing is used as a model to explore the advantages and problems deriving from the use of either enzymes or sugar binding proteins to develop stable fluorescence biosensors. We report on a novel approach to address the problem of substrate consumption by sensors based on enzymes, namely the utilization of apo-enzymes as non-active forms of the protein which are still able to bind the substrate/ligand. We also review studies in which derivatization of a naturally thermostable sugar-binding protein with a fluorescent probe allows quantitative monitoring of glucose binding even after immobilization on a solid support.     

Percutaneous fiber-optic sensor for chronic glucose monitoring in vivo

We are developing a family of fiber-optic sensors called Sencils (sensory cilia), which are disposable, minimally invasive, and can provide in vivo monitoring of various analytes for several weeks. The key element is a percutaneous optical fiber that permits reliable spectroscopic measurement of chemical reactions in a nano-engineered polymeric matrix attached to the implanted end of the fiber. This paper describes its first application to measure interstitial glucose based on changes in fluorescence resonance energy transfer (FRET) between fluorophores bound to betacyclodextrin and Concanavalin A (Con A) in a polyethylene glycol (PEG) matrix. In vitro experiments demonstrate a rapid and precise relationship between the ratio of the two fluorescent emissions and concentration of glucose in saline for the physiological range of concentrations (0-500mg/dl) over seven weeks. Chronic animal implantation studies have demonstrated good biocompatibility and durability for clinical applications.     

An evaluation of fluorescence polarization and lifetime discriminated polarization for high throughput screening of serine/threonine kinases

We used two kinases, c-jun N terminal kinase (JNK-1) and protein kinase C (PKC), as model enzymes to evaluate the potential of fluorescence polarization (FP) for high-throughput screening and the susceptibility of these assays to compound interference. For JNK-1 the enzyme kinetics in the FP assay were consistent with those found in a [gamma-33P]ATP filter wash assay. Determined pIC(50)s for nonfluorescent JNK-1 inhibitors were also consistent with those found in the filter wash assay. In contrast, fluorescent compounds were found to interfere with the JNK-1 FP assay, appearing as false positives, defined by their lack of activity in the filter wash assay. We also developed a second assay using a different kinase, protein kinase C, which was used to test a 5000 compound diversity set. As for JNK-1, interference from fluorescent compounds caused a high false positive rate. The Molecular Devices Corporation 'FLARe' instrument is capable of discriminating between fluorophores on the basis of their fluorescence (excited state) lifetime, and may assist in reducing compound interference in fluorescent assays. In both model FP kinase assays described here some, although not complete, reduction in interference from fluorescent compounds was achieved by the use of FLARe.     

G-protein-coupled receptor Gpr1 and G-protein Gpa2 of cAMP-dependent signaling pathway are involved in glucose-induced pexophagy in the yeast Saccharomyces cerevisiae

In yeast cell, glucose induces various changes of cellular metabolism on genetic and metabolic levels. One of such changes is autophagic degradation of dispensable peroxisomes (pexophagy) which occurs in vacuoles. We have found that in Saccharomyces cerevisiae, defect of G-protein-coupled receptor Gpr1 and G-protein Gpa2, both the components of cAMP-signaling pathway, strongly suppressed glucose-induced degradation of matrix peroxisomal protein thiolase. We conclude that proteins Gpr1 and Gpa2 are involved in glucose sensing and signal transduction during pexophagy process in yeast.     

Determination of optimal rhodamine fluorophore for in vivo optical imaging

Optical imaging has the potential to improve the efficacy of surgical and endoscopic approaches to cancer treatment; however, the optimal type of fluorescent probe has not yet been established. It is well-known that rhodamine-core-derived fluorophores offer a combination of desirable properties such as good photostability, high extinction coefficient, and high fluorescence quantum yield. However, despite the ubiquitous use of rhodamine fluorophores for in vivo optical imaging, it remains to be determined if unique chemical properties among individual rhodamine core family members affect fluorophore parameters critical to in vivo optical imaging applications. These parameters include preserved fluorescence intensity in low pH environments, similar to that of the endolysosome; efficient fluorescence signal despite conformational changes to targeting proteins as may occur in harsh subcellular environments; persistence of fluorescence after cellular internalization; and sufficient signal-to-background ratios to permit the identification of fluorophore-targeted tumors. In the present study, we conjugated 4 common rhodamine-core based fluorescent dyes to a clinically feasible and quickly internalizing D-galactose receptor targeting reagent, galactosamine serum albumin (GmSA), and conducted a series of in vitro and in vivo experiments using a metastatic ovarian cancer mouse model to determine if differences in optical imaging properties exist among rhodamine fluorophores and if so, which rhodamine core possesses optimal characteristics for in vivo imaging applications. Herein, we demonstrate that the rhodamine-fluorophore, TAMRA, is the most robust of the 4 common rhodamine fluorophores for in vivo optical imaging of ovarian cancer metastases to the peritoneum.     

Characterization of an on-line commercial fluorescence probe: modeling of the probe signal

A biosensor model was developed for a commercial NADH fluorescence probe to describe the single-frequency excitation and emission fluorescence behavior of an aqueous mixture of fluorophores. This model is essential in correlating the measured signals to the concentrations of fluorescent compounds in a bioreactor. In addition to the concentrations of fluorescent components, the relevant parameters of the model are the absorbance at both the excitation and the emission frequencies by the solvent and other absorbing species, the background signals, the light path length of the bioreactor vessel, the fluorescence yield, and the lampdetector configuration. Due to inner-filter effects and other interferences, the probe signal is intrinsically nonlinear in both the fluorophore concentration and the path length. An important parameter in the model is the geometric constant, S, which accounts for variations in the monitoring efficiency throughout the sample because fluorescent light is emitted in all directions. Previous models, derived from an unrealistic assumption that fluorescent light is emitted only in one direction parallel to the probe axis, are shown to be seriously deficient. The validity of the model was verified experimentally for a single-component solution in which both the fluorophore concentration and path length were varied.     

Observer-based online compensation of inner filter effect in monitoring fluorescence of GFP-expressing plant cell cultures

The green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria is a very useful reporter for real-time bioprocess sensing. GFP culture fluorescence is a composite signal that can be influenced by factors such as culture autofluorescence, inner filter effect (IFE), and photobleaching. These factors complicate accurate estimation of GFP concentrations from the culture fluorescence. IFE is especially problematic when using GFP in monitoring transgenic plant cell suspension cultures, due to the aggregated nature of the cells and the high biomass concentration in these culture systems. Reported approaches for online compensation of IFE in monitoring culture NADH fluorescence or bioluminescence require online measurement of biomass density or culture turbidity/optical density, in addition to fluorescence/bioluminescence measurement. In this study, culture GFP fluorescence was used successfully to estimate GFP concentration and other important states in bioreactor culture of transgenic tobacco cells, while the influences of IFE and culture autofluorescence were rectified without the need for an additional biomass sensor. This was achieved by setting up a novel model-based state observer. First, we developed an improved model for a backscatter fluorescence probe that takes into account the influence of IFE and autofluorescence on reporting culture GFP concentration from online fluorescence. The state observer was then established using the extended Kalman filter (EKF), based on the fluorescence probe model, a dynamic state model of the plant cell bioreactor, and online GFP fluorescence measurement. Several versions of the observer were introduced to address practical requirements associated with monitoring GFP fluorescence of plant cell cultures. The proposed approach offers an effective means for online compensation of IFE to enable quantitative interpretation of the culture fluorescence signals for accurate reporting of GFP or GFP-fusion protein expression.     

Position-specific incorporation of a highly photodurable and blue-laser excitable fluorescent amino acid into proteins for fluorescence sensing

A new fluorescent amino acid, L-2-acridonylalanine, was incorporated into proteins at specific positions using 4-base codon/anticodon strategy. The efficiency of the incorporation was high enough to obtain enough quantities of the mutants. The acridonyl group was highly fluorescent when it was excited at the wavelengths of blue-lasers and was highly photodurable compared with conventional fluorophores often used for biological analyses. The fluorescence intensity was sensitive to small changes in the polarity of the environment. When the nonnatural amino acid was incorporated into specific positions of streptavidin, the mutant protein worked as a fluorescent sensor to biotin. Similarly, when the amino acid was incorporated into camel single-chain antibody, the mutant protein sensitively responded to the antigen molecule. The high incorporation efficiency, the high photodurability, the excitability with blue-lasers, and high sensitivity to the environment make the acridonylalanine as the promising fluorescent amino acid for sensing small molecules when incorporated into specific positions of various antibodies, receptors, and enzymes.     

Photothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores

Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores.     

Imaging FRET between spectrally similar GFP molecules in single cells

Fluorescence resonance energy transfer (FRET) detection in fusion constructs consisting of green fluorescent protein (GFP) variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca(2+) ion release, and protease and kinase activity. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM). This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.