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David E Benson

Publications and source records attributed to David E Benson.

8 recordsLinked to original sources
PubMed2005

Selective, reversible, reagentless maltose biosensing with core-shell semiconducting nanoparticles.

Reagentless and reversible maltose biosensors are demonstrated using ZnS coated CdSe (CdSe@ZnS) nanoparticle emission intensities. This method is based on electron transfer quenching of unimolecular protein-CdSe@ZnS nanoparticle assemblies, which is provided by a protein-attached Ru(II) complex. This Ru(II) complex is presumed to reduce a valence band hole of the CdSe@ZnS excited state by tunneling through the ZnS overcoating. The Ru(II) complex mediated quenching of CdSe@ZnS nanoparticle emission was only decreased 1.2-fold relative to the CdSe nanoparticle systems. While four different Ru(II) complex attachment sites provided different amounts of nanoparticle emission quenching (1.20 to 1.75-fold decrease), all of these attachment sites yielded maltose-dependent intensity changes (1.1 to 1.4-fold increase upon maltose addition). Maltose dissociation constants for these four biosensing systems range from 250 nM to 1.0 microM, which are similar to the maltose-maltose binding protein dissociation constant that these sensors are based on. The increased fluorescence intensity was found to only occur in the presence of maltose. Furthermore, the ability of these reagentless protein-nanoparticle assemblies to perform maltose biosensing reversibly is demonstrated with the addition of alpha-glucosidase. Three 50 microM maltose additions after alpha-glucosidase addition showed increases of 2.2 microM, 600 nM, and 150 nM maltose. This result demonstrates a fluorometric method for examining alpha-glucosidase activity. Using maltose binding protein to control Ru(II) complex interactions with CdSe@ZnS nanoparticle surfaces provide a novel class of highly fluorescent, photostable biosensors that are selective for maltose.

Biosensing Techniques↗
PubMed2005

A modular nanoparticle-based system for reagentless small molecule biosensing.

Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.

Biosensing Techniques↗
PubMed2005

Magneto-structural relationships in a series of dinuclear oxalato-bridged (diphenyldipyrazolylmethane)copper(II) complexes.

A series of oxalato-bridged dinuclear copper(II) complexes of the general formula [Cu2(Pz2CPh2)2(X)2(mu-C2O4)] (X = Cl- (1), NO3(-) (2), ClO4(-) (3); Pz2CPh2 = diphenyldipyrazolylmethane) or [Cu2(Pz(3m)2CPh2)2(H2O)2(mu-C2O4)](NO3)2 x H2O (4) (Pz(3m)2CPh2 = diphenylbis(3-methylpyrazolyl)methane) was synthesized where the axial ligand was systematically varied to study its effect on structure and magnetic coupling. The structures of compounds 1, 2, and 4 have been elucidated by single-crystal X-ray diffraction. [Cu2(Pz2CPh2)2(Cl)2(mu-C2O4)] and [Cu2(Pz2CPh2)2(NO3)2(mu-C2O4)] are isostructural and crystallize in the triclinic system, space group P, Z = 2, with a = 8.6155(8) A, b = 10.1435(9) A, c = 11.3612(11) A, alpha = 95.535(2) degrees, beta = 110.303(2) degrees, and gamma = 106.111(2) degrees for 1 and with a = 8.863(7) A, b = 10.241(9) A, c = 11.425(10) A, alpha = 98.985(14) degrees, beta = 110.449(13) degrees, and gamma = 103.664(14) degrees for 2. [Cu2(Pz(3m)2CPh2)2(H2O)2(mu-C2O4)] x NO3 x H2O crystallizes in the monoclinic system, space group C2/c, Z = 4, with a = 23.4588(14) A, b = 8.8568(5) A, c = 21.7818(13) A, alpha = gamma = 90 degrees, and beta = 100.8890(10) degrees. Variable-temperature magnetic susceptibility studies indicate that all four compounds are strongly antiferromagnetically coupled (2J/k = -364, -344 cm(-1) (2), -424 cm(-1) (3), and -378 cm(-1) (4)). Magnetic and EPR results are discussed with respect to structural parameters to explore possible magneto-structural correlations.

Journal Article↗
PubMed2005

Geometric preferences of crosslinked protein-derived cofactors reveal a high propensity for near-sequence pairs.

Protein-derived cofactors that are composed of covalently crosslinked amino acid side chains are of increasing importance in protein science. These crosslinked protein-derived cofactors (CPDC) are formed either through direct oxidation by metal/O(2)-derived intermediates or through outer sphere oxidation by highly oxidizing cofactors. CPDCs that are formed by outer sphere oxidation do not require side-chain precursors to be coordinated by a metal center, and therefore are more difficult to identify than those formed by direct oxidation. To better understand the propensity for CPDC formation by outer sphere oxidation, the geometrical preferences of CPDCs were examined. The Dezymer algorithm has been used to identify all putative CPDC-forming mutations in 500 proteins. Geometrically, although chemically unrelated, these CPDCs were found to be similar to disulfide-bonded cysteine pairs. Additionally, the percentage of near-sequence pairs (i and i +1 to i and i + 5) increased as the average C(alpha)-C(alpha) distance between the amino acid pairs increased. This survey also examined the protein databank for proteins with pre-attack conformations for CPDCs, using non-bonded contacts reported by Procheck. A total of 323 unique proteins was identified, with 55 being near-sequence amino acid pairs. The high geometric propensity of near-sequence amino acid pairs for forming CPDCs is significant due to difficulties associated with detection by structural or mass spectrometric methods.

Base Sequence↗
PubMed2005

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.

Allosteric Site↗
PubMed2002

Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design.

Computational protein design methods were used to identify mutations that are predicted to introduce a binuclear copper center coordinated by six histidines, replacing the maltose-binding site in Escherichia coli maltose-binding protein (MBP) with an oxygen-binding site. A small family of five candidate designs consisting of 9 to 10 mutations each was constructed by oligonucleotide-directed mutagenesis. These mutant proteins were expressed and purified, and their stability, copper- and cobalt-binding properties, and interactions of the resulting metalloprotein complexes with azide, hydrogen peroxide, and dioxygen were characterized. We identified one 10-fold mutant, MBP.Hc.E, that can form Cu(II)(2) and Co(II)(2) complexes that interact with H(2)O(2) and O(2). The Co(II)(2) protein reacts with H(2)O(2) to form a complex that is spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core, whereas the Cu(II)(2) protein reacted with O(2) or H(2)O(2) does not. We postulate that the equilibrium between the open and closed conformations of MBP allows species with variable Cu-Cu distances to form, and that such species can bind ligands in geometries that are not observed in natural type III centers. Introduction of one additional mutation in the hinge region of MBP, I329F, known to favor formation of the closed state, results in a binuclear copper center that when reacted with low concentrations of H(2)O(2) mimics the spectroscopic signature of oxy-hemocyanin.

ATP-Binding Cassette Transporters↗
PubMed2001

Complex formation of cytochrome P450cam with Putidaredoxin. Evidence for protein-specific interactions involving the proximal thiolate ligand.

We have performed resonance Raman studies on ferrous NO- and CO-adducts of cytochrome P450(cam) and investigated the effects of diprotein complex formation with reduced putidaredoxin. We have found that the Fe-NO stretching mode of NO-P450(cam) can be resolved into two peaks at 551 and 561 cm(-1), and the binding of putidaredoxin increases the intensity of the high frequency component. Because the Fe-NO mode has been shown to be more sensitive to the nature of the heme proximal ligand than to the distal pocket environment, such a perturbation upon putidaredoxin binding is suggestive of changes in conformation or electronic structure that affect the proximal iron-cysteine bond. In accordance with this idea, the isotope shifts for the Fe-XO stretching and Fe-X-O bending modes (X = N or C) are insensitive to the presence or absence of putidaredoxin, indicating that the geometry of the Fe-X-O unit is not significantly altered by the complex formation. On the other hand, complex formation does induce a perturbation of the low frequency heme vibrational modes, suggesting that alterations of the heme electronic structure and/or geometry take place when putidaredoxin binds. We also find that cytochrome b(5) minimally affects the heme active site of the enzyme, although both putidaredoxin and cytochrome b(5) bind to the same or similar site on P450(cam). These observations suggest that there is a key specific interaction between P450(cam) and putidaredoxin, and that this interaction increases the population of a protein conformation that exhibits structural and/or electronic distortions of the heme group associated with the proximal side of the heme pocket and the S --> Fe electron donation. These electronic and structural changes are potentially correlated with H-bonding to the proximal cysteine.

Animals↗