Binding and signaling of surface-immobilized reagentless fluorescent biosensors derived from periplasmic binding proteins

Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Protein Science (Impact Factor: 2.85). 09/2006; 15(8):1936-44. DOI: 10.1110/ps.062261606
Source: PubMed


Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.

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    • "They have a theoretical capacity to detect any compound that can fit within the protein's binding pocket. Unfortunately, these computationally re-designed proteins are unstable in vitro, preventing use of this powerful technology in electronic detectors [3], [6]. These receptors have been used in bacterial biosensors, where re-designed PBPs were linked to gene expression through a histidine kinase signaling pathway [5]. "
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    ABSTRACT: There is an unmet need to monitor human and natural environments for substances that are intentionally or unintentionally introduced. A long-sought goal is to adapt plants to sense and respond to specific substances for use as environmental monitors. Computationally re-designed periplasmic binding proteins (PBPs) provide a means to design highly sensitive and specific ligand sensing capabilities in receptors. Input from these proteins can be linked to gene expression through histidine kinase (HK) mediated signaling. Components of HK signaling systems are evolutionarily conserved between bacteria and plants. We previously reported that in response to cytokinin-mediated HK activation in plants, the bacterial response regulator PhoB translocates to the nucleus and activates transcription. Also, we previously described a plant visual response system, the de-greening circuit, a threshold sensitive reporter system that produces a visual response which is remotely detectable and quantifiable. We describe assembly and function of a complete synthetic signal transduction pathway in plants that links input from computationally re-designed PBPs to a visual response. To sense extracellular ligands, we targeted the computational re-designed PBPs to the apoplast. PBPs bind the ligand and develop affinity for the extracellular domain of a chemotactic protein, Trg. We experimentally developed Trg fusions proteins, which bind the ligand-PBP complex, and activate intracellular PhoR, the HK cognate of PhoB. We then adapted Trg-PhoR fusions for function in plants showing that in the presence of an external ligand PhoB translocates to the nucleus and activates transcription. We linked this input to the de-greening circuit creating a detector plant. Our system is modular and PBPs can theoretically be designed to bind most small molecules. Hence our system, with improvements, may allow plants to serve as a simple and inexpensive means to monitor human surroundings for substances such as pollutants, explosives, or chemical agents.
    PLoS ONE 01/2011; 6(1):e16292. DOI:10.1371/journal.pone.0016292 · 3.23 Impact Factor
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    • "In the field of GBPs, there has been work in engineering of polypeptide sequences to enhance performance and accept various signal transduction markers (i.e., optical and electrochemical labels) [33] [34] [35] [36] [37] [38]. There have also been some reports in the literature on the immobilization of GBPs towards a glucose-sensing system [39]. However, to the best of the authors' knowledge, there have been no reports to date on the successful immobilization of GBPs or GBP-like sensing elements within a fixed matrix such that a continuous glucose-sensing device is realized. "
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    ABSTRACT: As the importance of blood-glucose control for both diabetic and non-diabetic patients continues to increase, there is a need for more advanced glucose-sensing technologies. In particular, an in vivo glucose sensor is needed that exhibits high accuracy when operating in a continuous manner for a relatively long period of time (3–5 days). Development of such sensors has been hampered, as low accuracy and sensor drift become major problems with in vivo environments, especially for enzyme-based electrochemical glucose sensors. This paper reports on the use of a novel, binding polypeptide-based, fluorescent, glucose-sensing system that promises to overcome many drawbacks of an enzyme-based system while showing the potential for high accuracy, especially at hypoglycemic levels.Fluorescently labeled glucose recognition polypeptide elements were immobilized in a polyacrylamide hydrogel matrix placed on the tip of an optical fiber to realize a continuous glucose-sensing device towards in vivo applications. In vitro validation was performed in both buffered solutions and whole blood to characterize sensor parameters such as sensitivity and response time. Testing demonstrated that the reagentless polypeptide-based glucose-sensing system has extreme sensitivity in the hypoglycemic levels while providing high precision across the entire human physiologic glucose range. Additionally, the sensor was shown to function at physiologic temperature (viz., 37 °C) and displayed high selectivity for glucose without interference from other sugars (viz., fructose).This represents the first report of implementing immobilized glucose binding protein-like elements in a sensing device for continuous glucose monitoring, and establishes proof-of-concept as an excellent alternative to overcoming problems of current long-term, continuous glucose-sensing technologies.
    Sensors and Actuators B Chemical 08/2010; 149(1-149):51-58. DOI:10.1016/j.snb.2010.06.031 · 4.10 Impact Factor
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    • "This can be used to link and spatially orient the biomolecule on the gold surface [15] [16] [17] [18] [19] [20] [21] [22]. When a surface-exposed cysteine residue on a protein is not feasible, thiol linkers to other residues on a protein allow semispecifically oriented layers on the surface [23]. Although there has been some discussion in the literature pointing to protein deposition without exposed cysteines, a number of studies from our laboratories have indicated that native glucose/galactose receptors (GGRs) without a genetically engineered cysteine residue do not bind to the gold surface, whereas receptors with a surface-exposed cysteine form a stable affinity bond to gold surfaces [15] [16] [17] [18] [19] [20] [21] [22]. "
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    ABSTRACT: Genetically engineered periplasmic glucose receptors as biomolecular recognition elements on gold nanoparticles (AuNPs) have allowed our laboratory to develop a sensitive and reagentless electrochemical glucose biosensor. The receptors were immobilized on AuNPs by a direct sulfur-gold bond through a cysteine residue that was engineered in position 1 on the protein sequence. The study of the attachment of genetically engineered and wild-type proteins binding to the AuNPs was first carried out in colloidal gold solutions. These constructs were studied and characterized by UV-Vis spectroscopy, transmission electron microscopy, particle size distribution, and zeta potential. We show that the genetically engineered cysteine is important for the immobilization of the protein to the AuNPs. Fabrication of the novel electrochemical biosensor for the detection of glucose used these receptor-coated AuNPs. The sensor showed selective detection of glucose in the micromolar concentration range, with a detection limit of 0.18 microM.
    Analytical Biochemistry 05/2008; 375(2):282-90. DOI:10.1016/j.ab.2007.12.035 · 2.22 Impact Factor
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