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.     

DEVELOPMENT OF GENETICALLY ENCODED FLUORESCENT PROTEIN CONSTRUCTS OF HYPERTHERMOPHILIC MALTOSE-BINDING PROTEIN

Circularly permuted green fluorescent protein (cGFP) was inserted into the hyperthermophilic maltose binding protein at two different locations. cGFP was inserted between amino acid residues 206 and 207, or fused to the N-terminal of maltose binding protein from Thermotoga maritima. The cloned DNA constructs were expressed in Escherichia coli cells, and purified by metal chelate affinity chromatography. Conformational change upon ligand binding was monitored by the increase in fluorescence intensity. Both of the fusion proteins developed significant fluorescence change at 0.5 mM maltose concentration, whereas their maltose binding affinities and optimum incubation times were different. Fluorescent biosensors based on mesophilic maltose binding proteins have been described in the literature, but there is a growing interest in biosensors based on thermostable proteins. Therefore, the developed protein constructs could be models for thermophilic protein-based fluorescent biosensors.     

Enzyme fluorescence as a sensing tool: new perspectives in biotechnology

The technology for fluorescence protein-sensing is advancing rapidly owing to the continued introduction of new concepts, new fluorophores, and proteins engineered for sensing-specific analytes. Concerns about the reversibility and selectivity of engineered proteins are being addressed by developing biosensors that are based on the utilisation of coenzyme-depleted enzymes. Such biomolecules do not consume the substrate and can exhibit conformational changes upon the binding of the analyte, which can be easily detected as fluorescence change. In addition, concerns about the stability of biosensors can be overcome by using thermostable enzymes isolated from thermophilic microorganisms. Finally, the development of new techniques such as polarization-based sensing, anisotropy-based sensing and lifetime-based sensing, all of which can be accomplished with light-emitting diodes as the light source, is prompting the design of a new class of specific and stable biosensors, as has occurred with blood glucose measurement. These biosensors represent a valid alternative to the conventional clinical chemistry diagnostics.     

Fluorescence sensing of intermolecular interactions and development of direct molecular biosensors

Molecular biosensors are devices of molecular size that are designed for sensing different analytes on the basis of biospecific recognition. They should provide two coupled functions - the recognition (specific binding) of the target and the transduction of information about the recognition event into a measurable signal. The present review highlights the achievements and prospects in design and operation of molecular biosensors for which the transduction mechanism is based on fluorescence. We focus on the general strategy of fluorescent molecular sensing, construction of sensor elements, based on natural and designed biopolymers (proteins and nucleic acids). Particular attention is given to the coupling of sensing elements with fluorescent reporter dyes and to the methods for producing efficient fluorescence responses.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Development of an optimized backbone of FRET biosensors for kinases and GTPases

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have shed new light on the spatiotemporal dynamics of signaling molecules. Among them, intramolecular FRET biosensors have been increasingly used due to their high sensitivity and user-friendliness. Time-consuming optimizations by trial and error, however, obstructed the development of intramolecular FRET biosensors. Here we report an optimized backbone for rapid development of highly sensitive intramolecular FRET biosensors. The key concept is to exclude the “orientation-dependent” FRET and to render the biosensors completely “distance-dependent” with a long, flexible linker. We optimized a pair of fluorescent proteins for distance-dependent biosensors, and then developed a long, flexible linker ranging from 116 to 244 amino acids in length, which reduced the basal FRET signal and thereby increased the gain of the FRET biosensors. Computational simulations provided insight into the mechanisms by which this optimized system was the rational strategy for intramolecular FRET biosensors. With this backbone system, we improved previously reported FRET biosensors of PKA, ERK, JNK, EGFR/Abl, Ras, and Rac1. Furthermore, this backbone enabled us to develop novel FRET biosensors for several kinases of RSK, S6K, Akt, and PKC and to perform quantitative evaluation of kinase inhibitors in living cells.     

Constructed molecular sensor to enhance metal detection by bacterial ribosomal switch-ion channel protein interaction

Molecular biosensors are useful tools that detect metal ions or other potentially toxic chemicals. However, the efficiency of conventional sensors is limited in mixed metals substrates, which is the common way they are found in nature. The use of biosensors constructed from genetically modified living microbial systems has the potential of providing sensitive detection systems for specific toxic targets. Consequently, our investigation was aimed at assembling different genetic building blocks to produce a focused microbial biosensor with the ability to detect specific metals. This objective was achieved by using a synthetic biology approach. Our genetic building blocks, including a synchronized ribosomal switch-iron ion channel, along with sequences of promoters, metal-binding proteins (Fe, Pb), ribosomal binding sites, yellow fluorescence reporter protein (YFRP), and terminators, were constructed within the same biobrick in Escherichia coli. We used an rpoS ribosomal switch containing an aptamer, which responds to the specific metal ligands, in synchronization with an iron ion channel, TonB. This switch significantly stimulates translation, as expressed by higher fluorescence, number of colonies, and concentration of RNA in E. coli. The positive results show the effectiveness of using genetically tailored synchronized ribosomal switch-ion channels to construct microbial biosensors to detect specific metals, as tested in iron solutions.     

Identification of cognate ligands for the Escherichia coli phnD protein product and engineering of a reagentless fluorescent biosensor for phosphonates

The Escherichia coli phnD gene is hypothesized to code for the periplasmic binding component of a phosphonate uptake system. Here we report the characterization of the phosphonate-binding properties of the phnD protein product. We find that PhnD exhibits high affinity for 2-aminoethylphosphonate (5 nM), the most commonly occurring natural phosphonate produced by lower eukaryotes, but also binds several other phosphonates with micromolar affinities. A significant number of man-made phosphonates, such as insecticides and chemical warfare agents, are chemical threats and environmental pollutants. Consequently, there is an interest in developing methods for the detection and bioremediation of phosphonates. Bacterial periplasmic-binding proteins have been utilized for developing reagentless biosensors that report analytes by coupling ligand-binding events to changes in the emission properties of a covalently conjugated environmentally-sensitive fluorophore. Several PhnD conjugates described here show large changes in fluorescence upon binding to methylphosphonate (MP), with two conjugates exhibiting up to 50% decrease in emission intensity. Since MP is the final degradation product of many nerve agents, these PhnD conjugates can function as components in a biosensor system for chemical warfare agents.     

Construction of a fluorescent biosensor family

Bacterial periplasmic binding proteins (bPBPs) are specific for a wide variety of small molecule ligands. bPBPs undergo a large, ligand-mediated conformational change that can be linked to reporter functions to monitor ligand concentrations. This mechanism provides the basis of a general system for engineering families of reagentless biosensors that share a common physical signal transduction functionality and detect many different analytes. We demonstrate the facility of designing optical biosensors based on fluorophore conjugates using 8 environmentally sensitive fluorophores and 11 bPBPs specific for diverse ligands, including sugars, amino acids, anions, cations, and dipeptides. Construction of reagentless fluorescent biosensors relies on identification of sites that undergo a local conformational change in concert with the global, ligand-mediated hinge-bending motion. Construction of cysteine mutations at these locations then permits site-specific coupling of environmentally sensitive fluorophores that report ligand binding as changes in fluorescence intensity. For 10 of the bPBPs presented in this study, the three-dimensional receptor structure was used to predict the location of reporter sites. In one case, a bPBP sensor specific for glutamic and aspartic acid was designed starting from genome sequence information and illustrates the potential for discovering novel binding functions in the microbial genosphere using bioinformatics.     

Ultrasensitive protein-DNA binding assays

Protein-DNA binding assays have been used in a variety of fields from fundamental studies regarding the binding process itself, to serving as probes for the detection, quantification and separation of target analytes. These assays have been used for the study of protein-DNA complex stoichiometry, the detection of DNA damage, and real-time separation of free and bound complexes by electrophoretic mobility. Synthetic DNA oligonucleotides, known as aptamers, have been increasingly used for affinity binding assays to proteins, as well as for separation studies and as biosensors. Recent advances have been made in protein-DNA binding assays using capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, molecular beacons, and affinity chromatography.     

Computational design and biochemical characterization of maize nonspecific lipid transfer protein variants for biosensor applications

Lipid transfer proteins (LTPs) are a family of proteins that bind and transfer lipids. Utilizing the maize LTP, we have successfully engineered fluorescent reagentless biosensors for the natural ligand of LTPs; this was achieved by using computational protein design to remove a disulfide bridge and attaching a thio-reactive fluorophore. Conformational change induced by ligand titration is thought to affect the fluorescence of the fluorophore, allowing detection of ligand binding. Fluorescence measurements show that our LTP variants have affinity to palmitate that is consistent with wild-type LTP. These molecules have the potential to be utilized as scaffolds to design hydrophobic ligand biosensors or to serve as drug carriers.     

Designed leucine‐rich repeat proteins bind two muramyl dipeptide ligands

Designed protein receptors hold diagnostic and therapeutic promise. We now report the design of five consensus leucine‐rich repeat proteins (CLRR4–8) based on the LRR domain of nucleotide‐binding oligomerization domain (NOD)‐like receptors involved in the innate immune system. The CLRRs bind muramyl dipeptide (MDP), a bacterial cell wall component, with micromolar affinity. The overall K _{d app} values ranged from 1.0 to 57 μM as measured by fluorescence quenching experiments. Biphasic fluorescence quenching curves were observed in all CLRRs, with higher affinity K _d1 values ranging from 0.04 to 4.5 μM, and lower affinity K _d2 values ranging from 3.1 to 227 μM. These biphasic binding curves, along with the docking studies of MDP binding to CLRR4, suggest that at least two MDPs bind to each protein. Previously, only single MDP binding was reported. This high‐capacity binding of MDP promises small, soluble, stable CLRR scaffolds as candidates for the future design of pathogen biosensors.     

A computational tool for designing FRET protein biosensors by rigid-body sampling of their conformational space

Biosensors relying on the fluorescence resonance energy transfer (FRET) between fluorescent proteins have been used for live-cell imaging of cellular events including Ca(2+) signaling. The efficiency of energy transfer between the donor and acceptor fluorescent proteins depends on the relative distance and orientation between them, which become altered by conformational changes of a fused sensory protein caused by a cellular event. In this way, changes in FRET efficiency of Ca(2+) biosensors can be correlated with Ca(2+) concentrations. The design of these FRET biosensors can be improved by modeling conformational changes before and after a cellular event. Hence, a computational tool called FPMOD was developed to predict FRET efficiency changes by constructing FRET biosensors and sampling their conformational space through rigid-body rotation. We showed with FPMOD that our computational modeling approach can qualitatively predict the FRET efficiencies of a range of biosensors, which had strong agreement with experimental results.     

Protein biosensors based on the principle of fluorescence resonance energy transfer for monitoring cellular dynamics

Genetically-coded, fluorescence resonance energy transfer (FRET) biosensors are widely used to study molecular events from single cells to whole organisms. They are unique among biosensors because of their spontaneous fluorescence and targeting specificity to both organelles and tissues. In this review, we discuss the theoretical basis of FRET with a focus on key parameters responsible for designing FRET biosensors that have the highest sensitivity. Next, we discuss recent applications that are grouped into four common biosensor design patterns—intermolecular FRET, intramolecular FRET, FRET from substrate cleavage and FRET using multiple colour fluorescent proteins. Lastly, we discuss recent progress in creating fluorescent proteins suitable for FRET purposes. Together these advances in the development of FRET biosensors are beginning to unravel the interconnected and intricate signalling processes as they are occurring in living cells and organisms.     

Ultrasensitive carbon nanotube-based biosensors using antibody-binding fragments

We report a method to build ultrasensitive carbon nanotube-based biosensors using immune binding reaction. Here carbon nanotube-field effect transistors (CNT-FETs) were functionalized with antibody-binding fragments as a receptor, and the binding event of target immunoglobulin G (IgG) onto the fragments was detected by monitoring the gating effect caused by the charges of the target IgG. Because the biosensors were used in buffer solution, it was crucial to use small-size receptors so that the charged target IgG could approach the CNT surface within the Debye length distance to give a large gating effect. The results show that CNT-FET biosensors using whole antibody had very low sensitivity (detection limit ∼1000 ng/ml), whereas those based on small Fab fragments could detect 1 pg/ml (∼7 fM level). Moreover, our Fab-modified CNT-FET could successfully block the nontarget proteins and could selectively detect the target protein in an environment similar to that of human serum electrolyte. Significantly, this strategy can be applied to general antibody-based detection schemes, and it should enable the production of label-free ultrasensitive electronic biosensors to detect clinically important biomarkers for disease diagnosis.     

Shining light on signaling and metabolic networks by genetically encoded biosensors

Fluorescent labels have revolutionized cell biology. Signaling intermediates and metabolites can be measured in real time with subcellular spatial resolution. Most of these sensors are based on fluorescent proteins, and many report fluorescence resonance energy transfer. Because the biosensors are genetically encoded, a toolbox for addressing cell biological questions at the systems level is now available. Fluorescent biosensors are able to determine the localization of proteins and their dynamics, to reveal the cellular and subcellular localization of the respective interactions and activities, and to provide complementary data on the steady state levels of ions, metabolites, and signaling intermediates with high temporal and spatial resolution. They represent the basis for cell-based high-throughput assays that are necessary for a systems perspective on plant cell function.     

Imaging local estrogen production in single living cells with recombinant fluorescent indicators

Estrogens are steroid hormones with many systemic effects in addition to development and maintenance of the female reproductive system, and ligands of estrogen receptors are of clinical importance because of their use as oral contraceptive, hormone replacement and antitumoral therapy. In addition, tumoral tissues have been found to express aromatase and other steroidogenic enzymes synthesizing estradiol. To aid in the understanding of these processes, we have developed assays to image the local production of estrogens in isolated living mammalian cells. We constructed biosensors based on estrogen receptor α ligand binding domain and fluorescent proteins by following two approaches. First, the ligand binding domain and a short fragment of steroid receptor coactivator-1 were appended to a circularly permuted yellow fluorescent protein to construct an excitation ratio estrogen indicator. In the second strategy, we constructed emission ratio sensors based on fluorescence resonance energy transfer, containing the ligand binding domain flanked by donor and acceptor fluorescent proteins. Estrogens altered the fluorescence signal of cells transfected with the indicators in a dose-dependent manner. We imaged local estrogen production in adrenocortical H295 cells expressing aromatase and transfected with the fluorescent sensors. In addition, paracrine detection was observed in HeLa cells harboring the indicators and co-cultured with H295 cells. This imaging approach may allow detection of physiological levels of these hormones in suitable animal models.     

Analysis of allosteric signal transduction mechanisms in an engineered fluorescent maltose biosensor

We previously reported the construction of a family of reagentless fluorescent biosensor proteins by the structure-based design of conjugation sites for a single, environmentally sensitive small molecule dye, thus providing a mechanism for the transduction of ligand-induced conformational changes into a macroscopic fluorescence observable. Here we investigate the microscopic mechanisms that may be responsible for the macroscopic fluorescent changes in such Fluorescent Allosteric Signal Transduction (FAST) proteins. As case studies, we selected three individual cysteine mutations (F92C, D95C, and S233C) of Escherichia coli maltose binding protein (MBP) covalently labeled with a single small molecule fluorescent probe, N-((2-iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), each giving rise to a robust FAST protein with a distinct maltose-dependent fluorescence response. The fluorescence emission intensity, anisotropy, lifetime, and iodide-dependent fluorescence quenching were determined for each conjugate in the presence and absence of maltose. Structure-derived solvent accessible surface areas of the three FAST proteins are consistent with experimentally observed quenching data. The D95C protein exhibits the largest fluorescence change upon maltose binding. This mutant was selected for further characterization, and residues surrounding the fluorophore coupling site were mutagenized. Analysis of the resulting mutant FAST proteins suggests that specific hydrogen-bonding interactions between the fluorophore molecule and two tyrosine side-chains, Tyr171 and Tyr176, in the open state but not the closed, are responsible for the dramatic fluorescence response of this construct. Taken together these results provide insights that can be used in future design cycles to construct fluorescent biosensors that optimize signaling by engineering specific hydrogen bonds between a fluorophore and protein.     

Comparative analysis of RNA/protein dynamics for the arginine-rich-binding motif and zinc-finger-binding motif proteins encoded by HIV-1

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.     

Circularly permuted monomeric red fluorescent proteins with new termini in the ��-sheet

Circularly permuted fluorescent proteins (FPs) have a growing number of uses in live cell fluorescence biosensing applications. Most notably, they enable the construction of single fluorescent protein‐based biosensors for Ca²⁺ and other analytes of interest. Circularly permuted FPs are also of great utility in the optimization of fluorescence resonance energy transfer (FRET)‐based biosensors by providing a means for varying the critical dipole–dipole orientation. We have previously reported on our efforts to create circularly permuted variants of a monomeric red FP (RFP) known as mCherry. In our previous work, we had identified six distinct locations within mCherry that tolerated the insertion of a short peptide sequence. Creation of circularly permuted variants with new termini at the locations corresponding to the sites of insertion led to the discovery of three permuted variants that retained no more than 18% of the brightness of mCherry. We now report the extensive directed evolution of the variant with new termini at position 193 of the protein sequence for improved fluorescent brightness. The resulting variant, known as cp193g7, has 61% of the intrinsic brightness of mCherry and was found to be highly tolerant of circular permutation at other locations within the sequence. We have exploited this property to engineer an expanded series of circularly permuted variants with new termini located along the length of the 10th β‐strand of mCherry. These new variants may ultimately prove useful for the creation of single FP‐based Ca²⁺ biosensors.