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Katie A. Edwards - One of the best experts on this subject based on the ideXlab platform.
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Periplasmic-Binding Protein-based biosensors and bioanalytical assay platforms: Advances, considerations, and strategies for optimal utility
'Elsevier BV', 2021Co-Authors: Katie A. EdwardsAbstract:Periplasmic Binding Proteins provide gram-negative bacteria with mechanisms for nutrient uptake and sensing their environments through recognition and transport of small molecules and ions. In the assay development realm, the biosensing niche that these Proteins fulfill is the recognition and detection of non-immunogenic targets, including inorganic ions, vitamins, amino acids, and sugars with affinities in the low nM to low μM range. These Proteins have been applied in a variety of homogeneous and heterogeneous platforms including those based on enzyme-linked immunosorbent assays, surface plasmon resonance, immunomagnetic separation, and fluorescence resonance energy transfer. Their Binding repertoire is largely limited to their natural targets, although progress has been made in engineering the target specificity by grafting alternate Binding pockets and understanding the stability limitations inherent in engineering Proteins for non-native targets. There has been significant interest in the glucose-galactose Binding Protein as an alternative to enzymes for diabetic glucose monitoring, but emerging applications for these Proteins include environmental and microbiological sensing. Within, a comprehensive review of sensor advances is presented from a perspective of assay design, engineering challenges, and practical application. Immobilization strategies, labeling considerations, and overcoming stoichiometric signal limitations to yield optimal performance are discussed
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Periplasmic Binding Protein based magnetic isolation and detection of thiamine in complex biological matrices
Talanta, 2019Co-Authors: Katie A. Edwards, Seth Feder, Eileen A Randall, Nicole Tumaung, David R Sannino, Esther R Angert, Clifford E. KraftAbstract:Abstract Deficiencies in thiamine (vitamin B1) cause a host of neurological and reproductive impairments yielding morbidity and mortality across environmental and clinical realms. In a technique analogous to immunomagnetic separation, we introduce the use of thiamine Periplasmic Binding Protein (TBP)-conjugated magnetic beads to isolate thiamine from complex matrices. TBP expressed in Escherichia coli is highly specific to thiamine and provides an alternative to antibodies for this non-immunogenic target. After incubation with the sample and removal of unbound matrix constituents, thiamine is simultaneously released and converted to its fluorescent oxidation product thiochrome by alkaline potassium ferricyanide. Subsequent measurement of fluorescence at thiochrome-specific wavelengths provides a second layer of specificity for the detection of thiamine. Thiamine could be quantified at concentrations as low as 5 nM ranging up to 240 nM. Within, we apply this technique to selectively capture and quantify thiamine in complex salmonid fish egg and tissue matrices. Our results showed no measurable non-specific Binding to the beads by endogenous fluorophores in the fish egg matrix. Thiamine levels as low as 0.2 nmol/g of fish egg can be detected using this approach, which is sufficient to assess deficiencies causing morbidity and mortality in fish that occur at 1.0 nmol/g of egg. This practical method may find application in other resource limited settings for clinical, food, or dietary supplement analyses.
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High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition.
Analytical chemistry, 2016Co-Authors: Katie A. Edwards, Woo Jin Seog, Lu Han, Seth Feder, Clifford E. Kraft, Antje J. BaeumnerAbstract:Although antibodies and aptamers are commonly used bioaffinity recognition elements, they are not available for many important analytes. As an alternative, we demonstrate use of a Periplasmic Binding Protein (PBP) to provide high affinity recognition for thiamine (vitamin B1), an analyte of great importance to human and environmental health for which, like so many other small molecules, no suitable biorecognition element is available. We demonstrate that with an appropriate competitive strategy, a highly sensitive (limit of detection of 0.5 nM) and specific bioassay for thiamine and its phosphorylated derivatives can be designed. The high-throughput method relies upon the thiamine Periplasmic Binding Protein (TBP) from Escherichia coli for thiamine biorecognition and dye-encapsulating liposomes for signal-enhancement. A thiamine monosuccinate-PEG-biotin derivative was synthesized to serve as an immobilized competitor that overcame constraints imposed by the deep Binding cleft and structural recognition re...
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Periplasmic Binding Protein based detection of maltose using liposomes a new class of biorecognition elements in competitive assays
Analytical Chemistry, 2013Co-Authors: Katie A. Edwards, Antje J. BaeumnerAbstract:A Periplasmic Binding Protein (PBP) was investigated as a novel Binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10–4 M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these Proteins’ two globular domains upon target Binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose Binding Protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the Protein. MBP con...
Timothy A Mietzner - One of the best experts on this subject based on the ideXlab platform.
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the ferric iron Binding Protein of pathogenic neisseria spp functions as a Periplasmic transport Protein in iron acquisition from human transferrin
Molecular Microbiology, 1993Co-Authors: Chengyen Chen, Sally A Berish, Stephen A Morse, Timothy A MietznerAbstract:Summary The ferric iron-Binding Protein (Fbp) expressed by pathogenic Neisseria spp. has been proposed to play a central role in the high-affinity acquisition of iron from human transferrin. The results of this investigation provide evidence that Fbp participates in this process as a functional analogue of a Gram-negative Periplasmic-Binding Protein component, which operates as a part of a general active transport process for the receptor-mediated, high-affinity transport of iron from human transferrin. Known properties of Fbp are correlated with those of other well-characterized Periplasmic-Binding Proteins, including structural features and the reversible Binding of ligand. Predictive of a Periplasmic-Binding Protein, which functions in the high-affinity acquisition of iron, is that Fbp is a transient participant in the process of iron acquisition from human transferrin. Evidence for this is demonstrated by results of pulse–chase experiments. Taken together, the data described here and elsewhere suggest that pathogenic Neisseria spp. use a Periplasmic-Binding Protein-mediated active transport mechanism for the acquisition of iron from human transferrin.
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the ferric iron Binding Protein of pathogenic neisseria spp functions as a Periplasmic transport Protein in iron acquisition from human transferrin
Molecular Microbiology, 1993Co-Authors: Chengyen Chen, Sally A Berish, Stephen A Morse, Timothy A MietznerAbstract:The ferric iron-Binding Protein (Fbp) expressed by pathogenic Neisseria spp. has been proposed to play a central role in the high-affinity acquisition of iron from human transferrin. The results of this investigation provide evidence that Fbp participates in this process as a functional analogue of a Gram-negative Periplasmic-Binding Protein component, which operates as a part of a general active transport process for the receptor-mediated, high-affinity transport of iron from human transferrin. Known properties of Fbp are correlated with those of other well-characterized Periplasmic-Binding Proteins, including structural features and the reversible Binding of ligand. Predictive of a Periplasmic-Binding Protein, which functions in the high-affinity acquisition of iron, is that Fbp is a transient participant in the process of iron acquisition from human transferrin. Evidence for this is demonstrated by results of pulse-chase experiments. Taken together, the data described here and elsewhere suggest that pathogenic Neisseria spp. use a Periplasmic-Binding Protein-mediated active transport mechanism for the acquisition of iron from human transferrin.
Jonathan D Dattelbaum - One of the best experts on this subject based on the ideXlab platform.
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engineering a switch based biosensor for arginine using a thermotoga maritima Periplasmic Binding Protein
Analytical Biochemistry, 2017Co-Authors: Teraya Donaldson, Alessio Ausili, Sabato Dauria, Luisa Iozzino, Lindsay J Deacon, Hilbert Billones, Jonathan D DattelbaumAbstract:The Thermotoga maritima arginine-Binding Protein (TmArgBP) has been modified to create a reagentless fluorescent Protein biosensor. Two design methods for biosensor construction are compared: 1) solvent accessibility of environmentally-sensitive probes and 2) fluorescence deactivation due to photo-induced electron transfer (PET). Nine single cysteine TmArgBP mutants were created and labeled with three different environmentally sensitive fluorescent probes. These mutants demonstrated limited changes in fluorescence emission upon the addition of arginine. In contrast, the PET-based biosensor provides significant enhancements over the traditional approach and provides a fluorescence quenching mechanism that was capable of providing quantitative detection of arginine. Site-directed mutagenesis of TmArgBP was used to create attachment points for the fluorescent probe (K145C) and for an internal aromatic residue (D18X) to serve as the PET quencher. Both tyrosine and tryptophan, but not phenylalanine, were able to quench the emission of the fluorescent probe by more than 80% upon the addition of arginine. The dissociation constant for arginine ranged from 0.87 to 1.5 μM across the different sensors. This PET-based strategy provides a simple and broadly applicable approach for the analytical detection of small molecules that may be applied to any Protein that exhibits conformational switching in a ligand dependent manner.
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tryptophan scanning mutagenesis of the ligand Binding pocket in thermotoga maritima arginine Binding Protein
Biochimie, 2014Co-Authors: Lindsay J Deacon, Anna Pennacchio, Sabato Dauria, Teraya Donaldson, Luisa Iozzino, Hilbert Billones, Anne A Galyean, Jonathan D DattelbaumAbstract:Abstract The Thermotoga maritima arginine Binding Protein (TmArgBP) is a member of the Periplasmic Binding Protein superfamily. As a highly thermostable Protein, TmArgBP has been investigated for the potential to serve as a Protein scaffold for the development of fluorescent Protein biosensors. To establish a relationship between structural dynamics and ligand Binding capabilities, we constructed single tryptophan mutants to probe the arginine Binding pocket. Trp residues placed around the Binding pocket reveal a strong dependence on fluorescence emission of the Protein with arginine for all but one of the mutants. Using these data, we calculated dissociation constants of 1.9–3.3 μM for arginine. Stern–Volmer quenching analysis demonstrated that the Protein undergoes a large conformational change upon ligand Binding, which is a common feature of this Protein superfamily. While still active at room temperature, time-resolved intensity and anisotropy decay data suggest that the Protein exists as a highly rigid structure under these conditions. Interestingly, TmArgBP exists as a dimer at room temperature in both the presence and absence of arginine, as determined by asymmetric flow field flow fractionation (AF4) and supported by native gel-electrophoresis and time-resolved anisotropy. Our data on dynamics and stability will contribute to our understanding of hyperthermophilic Proteins and their potential biotechnological applications.
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amino acid transport in thermophiles characterization of an arginine Binding Protein from thermotoga maritima 3 conformational dynamics and stability
Journal of Photochemistry and Photobiology B-biology, 2013Co-Authors: Alessio Ausili, Anna Pennacchio, Maria Staiano, Jonathan D Dattelbaum, Dimitrios Fessas, Alberto Schiraldi, Sabato DauriaAbstract:Abstract Arginine-Binding Protein from Thermotoga maritima (TmArgBP) is a 27.7 kDa Protein possessing the typical two domain structure of the Periplasmic Binding Protein family. The Protein is characterized by high specificity and affinity for Binding a single molecule of l -arginine. In this work, the effect of temperature and/or guanidine hydrochloride on structure and stability of the Protein in the absence and in the presence of l -arginine has been investigated by differential scanning calorimetry, far-UV circular dichroism and intrinsic tryptophan phosphorescence and fluorescence. The results revealed that TmArgBP undergoes an irreversible one-step thermal unfolding process in a cooperative mode. The TmArgBP melting temperature was recorded at 115 °C. The presence of l -arginine did not change the Protein secondary structure content as well as the intrinsic phosphorescence and fluorescence Protein properties, even if it increases the structural stability of the Protein. The obtained results are discussed in combination with a detailed inspection of the three-dimensional structure of the Protein.
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amino acid transport in thermophiles characterization of an arginine Binding Protein in thermotoga maritima
Molecular BioSystems, 2009Co-Authors: Matthew S Luchansky, Sabato Dauria, Luisa Iozzino, Gabriella Pocsfalvi, Daniela Marasco, Jonathan D DattelbaumAbstract:Members of the Periplasmic Binding Protein superfamily are involved in the selective passage of ligands through bacterial cell membranes. The hyperthermophilic eubacterium Thermotoga maritima was found to encode a highly stable and specific Periplasmic arginine-Binding Protein (TM0593). Following signal sequence removal and overexpression in Escherichia coli, TM0593 was purified by thermoprecipitation and affinity chromatography. The ultra-stable Protein with a monomeric molecular weight of 27.7 kDa was found to exist as both a homodimer and homotrimer at appreciable concentrations even under strongly denaturing conditions, with an estimated transition temperature of 116 °C. Its multimeric structure may provide further evidence of the importance of quaternary structure in the movement of nutrients across bacterial membranes. Purified and refolded TM0593 was further characterized by fluorescence spectroscopy, mass spectrometry, and circular dichroism to demonstrate the specificity of the Protein for arginine and to elucidate structural changes associated with arginine Binding. The Protein binds arginine with a dissociation constant of 20 μM as determined by surface plasmon resonance measurements. Due to its high thermodynamic stability, TM0593 may serve as a scaffold for the creation of a robust fluorescent biosensor.
Antje J. Baeumner - One of the best experts on this subject based on the ideXlab platform.
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High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition.
Analytical chemistry, 2016Co-Authors: Katie A. Edwards, Woo Jin Seog, Lu Han, Seth Feder, Clifford E. Kraft, Antje J. BaeumnerAbstract:Although antibodies and aptamers are commonly used bioaffinity recognition elements, they are not available for many important analytes. As an alternative, we demonstrate use of a Periplasmic Binding Protein (PBP) to provide high affinity recognition for thiamine (vitamin B1), an analyte of great importance to human and environmental health for which, like so many other small molecules, no suitable biorecognition element is available. We demonstrate that with an appropriate competitive strategy, a highly sensitive (limit of detection of 0.5 nM) and specific bioassay for thiamine and its phosphorylated derivatives can be designed. The high-throughput method relies upon the thiamine Periplasmic Binding Protein (TBP) from Escherichia coli for thiamine biorecognition and dye-encapsulating liposomes for signal-enhancement. A thiamine monosuccinate-PEG-biotin derivative was synthesized to serve as an immobilized competitor that overcame constraints imposed by the deep Binding cleft and structural recognition re...
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High-Throughput Detection of Thiamine Using Periplasmic Binding Protein-Based Biorecognition
2016Co-Authors: Katie A. Edwards, Woo Jin Seog, Lu Han, Seth Feder, Clifford E. Kraft, Antje J. BaeumnerAbstract:Although antibodies and aptamers are commonly used bioaffinity recognition elements, they are not available for many important analytes. As an alternative, we demonstrate use of a Periplasmic Binding Protein (PBP) to provide high affinity recognition for thiamine (vitamin B1), an analyte of great importance to human and environmental health for which, like so many other small molecules, no suitable biorecognition element is available. We demonstrate that with an appropriate competitive strategy, a highly sensitive (limit of detection of 0.5 nM) and specific bioassay for thiamine and its phosphorylated derivatives can be designed. The high-throughput method relies upon the thiamine Periplasmic Binding Protein (TBP) from Escherichia coli for thiamine biorecognition and dye-encapsulating liposomes for signal-enhancement. A thiamine monosuccinate-PEG-biotin derivative was synthesized to serve as an immobilized competitor that overcame constraints imposed by the deep Binding cleft and structural recognition requirements of PBPs. The assay was applied to ambient environmental samples with high reproducibility. These findings demonstrate that PBPs can serve as highly specific and sensitive affinity recognition elements in bioanalytical assay formats, thereby opening up the field of affinity sensors to a new range of analytes
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Periplasmic Binding Protein based detection of maltose using liposomes a new class of biorecognition elements in competitive assays
Analytical Chemistry, 2013Co-Authors: Katie A. Edwards, Antje J. BaeumnerAbstract:A Periplasmic Binding Protein (PBP) was investigated as a novel Binding species in a similar manner to an antibody in a competitive enzyme linked immunosorbent assay (ELISA), resulting in a highly sensitive and specific assay utilizing liposome-based signal amplification. PBPs are located at high concentrations (10–4 M) between the inner and outer membranes of gram negative bacteria and are involved in the uptake of solutes and chemotaxis of bacteria toward nutrient sources. Previous sensors relying on PBPs took advantage of the change in local environment or proximity of site-specific fluorophore labels resulting from the significant conformational shift of these Proteins’ two globular domains upon target Binding. Here, rather than monitoring conformational shifts, we have instead utilized the maltose Binding Protein (MBP) in lieu of an antibody in an ELISA. To our knowledge, this is the first PBP-based sensor without the requirement for engineering site-specific modifications within the Protein. MBP con...
David J Kelly - One of the best experts on this subject based on the ideXlab platform.
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a new mechanism for high affinity uptake of c4 dicarboxylates in bacteria revealed by the structure of rhodopseudomonas palustris matc rpa3494 a Periplasmic Binding Protein of the tripartite tricarboxylate transporter ttt family
Journal of Molecular Biology, 2018Co-Authors: Leonardo T Rosa, John B Rafferty, David J KellyAbstract:Abstract C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute Binding Protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the Periplasmic Binding Protein from this system, binds to l - and d -malate with Kd values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (Kd = 110 nM) and fumarate (Kd = 400 nM). The 2.1-A crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the Protein structure. The substrate coordination in MatC correlates with the Binding data and explains the Protein's selectivity for different substrates and respective Binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure–function relationships and transport mechanisms have been lacking.
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the coupstu and tarpqm transporters in rhodopseudomonas palustris redundant promiscuous uptake systems for lignin derived aromatic substrates
PLOS ONE, 2013Co-Authors: Robert C Salmon, Matthew J Cliff, John B Rafferty, David J KellyAbstract:The biodegradation of lignin, one of the most abundant carbon compounds on Earth, has important biotechnological applications in the derivation of useful products from lignocellulosic wastes. The purple photosynthetic bacterium Rhodopseudomonas palustris is able to grow photoheterotrophically under anaerobic conditions on a range of phenylpropeneoid lignin monomers, including coumarate, ferulate, caffeate, and cinnamate. RPA1789 (CouP) is the Periplasmic Binding-Protein component of an ABC system (CouPSTU; RPA1789, RPA1791–1793), which has previously been implicated in the active transport of this class of aromatic substrate. Here, we show using both intrinsic tryptophan fluorescence and isothermal titration calorimetry that CouP binds a range of phenylpropeneoid ligands with Kd values in the nanomolar range. The crystal structure of CouP with ferulate as the bound ligand shows H-bond interactions between the 4-OH group of the aromatic ring with His309 and Gln305. H-bonds are also made between the carboxyl group on the ferulate side chain and Arg197, Ser222, and Thr102. An additional transport system (TarPQM; RPA1782–1784), a member of the tripartite ATP-independent Periplasmic (TRAP) transporter family, is encoded at the same locus as rpa1789 and several other genes involved in coumarate metabolism. We show that the Periplasmic Binding-Protein of this system (TarP; RPA1782) also binds coumarate, ferulate, caffeate, and cinnamate with nanomolar Kd values. Thus, we conclude that R. palustris uses two redundant but energetically distinct primary and secondary transporters that both employ high-affinity Periplasmic Binding-Proteins to maximise the uptake of lignin-derived aromatic substrates from the environment. Our data provide a detailed thermodynamic and structural basis for understanding the interaction of lignin-derived aromatic substrates with Proteins and will be of use in the further exploitation of the flexible metabolism of R. palustris for anaerobic aromatic biotransformations.
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topological analysis of dctq the small integral membrane Protein of the c4 dicarboxylate trap transporter of rhodobacter capsulatus
Fems Microbiology Letters, 2001Co-Authors: Neil R Wyborn, Jesse Alderson, Simon C Andrews, David J KellyAbstract:Tripartite ATP-independent Periplasmic (‘TRAP’) transporters are a novel group of bacterial and archaeal secondary solute uptake systems which possess a Periplasmic Binding Protein, but which are unrelated to ATP-Binding cassette (ABC) systems. In addition to the Binding Protein, TRAP transporters contain two integral membrane Proteins or domains, one of which is 40–50 kDa with 12 predicted transmembrane (TM) helices, thought to be the solute import Protein, while the other is 20–30 kDa and of unknown function. Using a series of plasmid-encoded β-lactamase fusions, we have determined the topology of DctQ, the smaller integral membrane Protein from the high-affinity C4-dicarboxylate transporter of Rhodobacter capsulatus, which to date is the most extensively characterised TRAP transporter. DctQ was predicted by several topology prediction programmes to have four TM helices with the N- and C-termini located in the cytoplasm. The levels of ampicillin resistance conferred by the fusions when expressed in Escherichia coli were found to correlate with this predicted topology. The data have provided a topological model which can be used to test hypotheses concerning the function of the different regions of DctQ and which can be applied to other members of the DctQ family.
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purification characterization and nucleotide sequence of the Periplasmic c4 dicarboxylate Binding Protein dctp from rhodobacter capsulatus
Molecular Microbiology, 1991Co-Authors: Jonathan G Shaw, Mark J Hamblin, David J KellyAbstract:: A Periplasmic Binding Protein essential for high-affinity transport of the C4-dicarboxylates malate, succinate and fumarate across the cytoplasmic membrane of the purple photosynthetic bacterium Rhodobacter capsulatus has been purified to homogeneity and some of its ligand-Binding properties characterized. The Protein was not produced in a Tn5 insertion mutant unable to transport C4-dicarboxylates under aerobic conditions in the dark. Wild-type DNA corresponding to the location of the transposon insertion site was subcloned and a 1.5 kb section sequenced. A complete open reading frame of 999 bp was identified that encoded a 333-residue Protein (DctP) with a molecular weight of 36,128 with a 26-residue amino-terminal signal peptide. The identify of this Protein with the purified dicarboxylate-Binding Protein and the position of the predicted signal peptide cleavage site was confirmed by N-terminal sequencing. No significant homology with other Proteins was detected in database searches. A GC-rich region of dyad symmetry was located 7 bp downstream of the dctP translational stop codon. This structure may be of significance in regulating the relative abundance of DctP and other dct gene products which comprise the high-affinity dicarboxylate transport system in this bacterium.