A single amino acid Gly-tag enables metal-free protein purification

Analytically pure proteins are indispensable for diverse applications, including therapeutics. Here, we report a methodology where a single amino acid, glycine, enables metal-free protein purification. This robust platform is enabled by a Gly-tag resin for site-specific capture, enrichment, and release through chemically triggered C–C bond dissociation by resonance-assisted electron density polarization.

Gly-tag resin precisely captures and releases a protein with one glycine at the N-terminus. The user-friendly protocol delivers analytically pure protein free of metal contaminants.     

Effect of pharmaceuticals on thermoreversible gelation of PEO-PPO-PEO copolymers

Pluronic F127, a triblock copolymer of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO), has generated considerable interest as a drug delivery vehicle due to its ability to gel at physiological temperatures. This work examines the gelation behavior of Pluronic F127 in the presence of a series of hydrophobic pharmaceuticals, to determine whether there is any correlation between gelation and physicochemical parameters of drug solutes. The study includes the local anesthetics dibucaine, lidocaine, and tetracaine; the pharmaceutical additives methyl paraben, ethyl paraben, and propyl paraben; the anti-cancer agents paclitaxel and baccatin III; and the anti-inflammatory agent sulindac. The results indicate that the presence of local anesthetics and pharmaceutical additives allows F127 solutions to form gels at lower copolymer concentrations; local anesthetics and pharmaceutical additives also shift gelation down to a lower gelation temperature. This behavior is strongly dependent on drug solubility; poorly soluble drugs (paclitaxel, baccatin III, sulindac) do not change the lower gelation temperature or minimum F127 concentration for gelation. An equation relating the decrease in gelation temperature to drug solubility is presented, and the equation fits the data well. The results have significant positive implications on the toxicity and economic issues related to use of Pluronic F127 in drug delivery.     

Nickel(II)-Mediated Reversible Thiolate/Disulfide Conversion as a Mimic for a Key Step of the Catalytic Cycle of Methyl-Coenzyme M Reductase

According to the well-accepted mechanism, methyl-coenzyme M reductase (MCR) involves Ni-mediated thiolate-to-disulfide conversion that sustains its catalytic cycle of methane formation in the energy saving pathways of methanotrophic microbes. Model complexes that illustrate Ni-ion mediated reversible thiolate/disulfide transformation are unknown. In this paper we report the synthesis, crystal structure, spectroscopic properties and redox interconversions of a set of Ni^II complexes comprising a tridentate N₂S donor thiol and its analogous N₄S₂ donor disulfide ligands. These complexes demonstrate reversible Ni^II-thiolate/Ni^II-disulfide (both bound and unbound disulfide-S to Ni^II) transformations via thiyl and disulfide monoradical anions that resemble a primary step of MCR's catalytic cycle.     

Eco-friendly synthesis of jasminaldehyde by condensation of 1-heptanal with benzaldehyde using hydrotalcite as a solid base catalyst

The catalytic activity of hydrotalcite ([M(II)_1−xM(III)_x(OH₂)]^x+(CO₃²⁻)_x/n·mH₂O; where M(II) = Mg, Ni, Zn and M(III) = Al) was evaluated for the synthesis of jasminaldehyde by solvent free condensation of 1-heptanal with benzaldehyde. The effect of activation of as-synthesized Mg-Al hydrotalcite samples of varied Mg/Al molar ratio on its catalytic activity was studied and correlated with their basicity as determined from the model test reaction. The effect of reaction parameters such as, amount of catalyst, benzaldehyde to 1-heptanal molar ratio and reaction temperature on conversion of 1-heptanal and selectivity of jasminaldehyde was studied in detail. Maximum selectivity of jasminaldehyde (86%) with 98% conversion of 1-heptanal was observed using as-synthesized Mg-Al hydrotalcite of Mg/Al molar ratio of 3.5 as a catalyst. The kinetics of the reaction was measured and reaction rate and order of reaction were determined under optimum reaction conditions. The catalyst was re-used upto three cycles without significant loss in its activity. The base catalyzed reaction mechanism for condensation of 1-heptanal with benzaldehyde is proposed.     

Preparation, XRD and Raman spectroscopic studies on new compounds RE2Hf2O7 (RE=Dy, Ho, Er, Tm, Lu, Y): Pyrochlores or defect-fluorite?

A series of compositions with the general formula RE₂Hf₂O₇ (RE=Dy, Ho, Er, Tm, Y and Lu) was prepared by a standard solid-state route and characterized by powder X-ray diffraction (XRD) and Raman spectroscopy. As per theoretical modeling reported in literature, some of these materials were predicted to exist in pyrochlore lattice. However, a careful X-ray diffraction, Raman spectroscopic and synchrotron radiation-XRD study revealed that under the experimental conditions used in the present investigation, out of all the RE₂Hf₂O₇ samples only Dy₂Hf₂O₇ has got a tendency to form a pyrochlore structure. All the other (Ho, Er, Tm, Lu, Y) hafnates crystallize in a defect-fluorite structure. In order to further ascertain these inferences, a few more RE₂Hf₂O₇ samples (La, Nd, Sm) i.e., with larger RE³⁺ ions were also prepared and the results were compared.     

Creatinine sensor based on a molecularly imprinted polymer-modified hanging mercury drop electrode

Molecularly imprinted polymers (MIP) have been elucidated to work as artificial receptors. In our present study, a MIP was applied as a molecular recognition element to a chemical sensor. We have constructed a creatinine sensor based on a MIP layer selective for creatinine and its differential pulse, cathodic stripping voltammetric detection (DPCSV) on a hanging mercury drop electrode (HMDE). The creatinine sensor was fabricated by the drop coating of dimethylformamide (DMF) solution of a creatinine-imprinted polymer onto the surface of HMDE. The modified-HMDE, preanodised in neutral medium at +0.4 V versus Ag/AgCl for 120 s, exhibited a marked enhancement in DPCSV current in comparison to the less anodised (≤+0.3 V) HMDE. The creatinine was preconcentrated and instantaneously oxidised in MIP layer giving DPCSV response in the concentration range of 0.0025-84.0 μg mL⁻¹ [detection limit (3σ) 1.49 ng mL⁻¹]. The sensor was found to be highly selective for creatinine without any response of interferents viz., NaCl, urea, creatine, glucose, phenylalanine, tyrosine, histidine and cytosine. The non-imprinted polymer-modified electrode did not show linear response to creatinine. The imprinting factor as high as 9.4 implies that the imprinted polymer exclusively acts as a recognition element of creatinine sensor. The proposed procedure can be used to determine creatinine in human blood serum without any preliminary treatment of the sample in an accurate, rapid and simple way.     

Thermodynamics of activated phase transitions of 8CB: DSC and MC calorimetry

The present paper reports the heating rate effect on the phase transitions of a pure liquid crystal octylcyanobiphenyl (8CB) with use of Differential Scanning Calorimetry (DSC) and Modulation Calorimetry (MC) techniques. The DSC runs were taken at various temperature ramp rates from 20 to 0.5 K/min for heating and cooling scans. Well-defined endothermic/exothermic peaks were found at the melting/crystallization, smectic-A to nematic (SmA-N), and nematic to isotropic (N-I) transitions on heating/cooling scans, respectively. All transitions shift in temperature significantly with different ramp rates. The temperature shift of C(p) peaks between heating and cooling scans indicates the order of the transitions. In addition, all transitions follow an Arrhenius behavior. The activation energy of a transition increases as the total energy involved in the transition decreases. The respective enthalpy and entropy change of each transition provides information on the Gibbs free energy. The significance of the results is discussed in terms of the order of transitions. A comparative analysis of MC and DSC techniques highlights the significance of the two techniques. MC is a practicable tool for observing the phase dynamics whereas DSC is a good tool for studying the rate kinematics of the transitions.     

Dioxygenase-catalysed oxidation of disubstituted benzene substrates: benzylic monohydroxylation versus aryl cis-dihydroxylation and the meta effect

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric substituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations.     

Functional genetic analysis reveals a 2-Alkyl-4-quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria

Pseudomonas aeruginosa synthesizes diverse 2-alkyl-4(1H)-quinolones (AHQs), including the signaling molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), via the pqsABCDE locus. By examining the genome databases, homologs of the pqs genes were identified in other bacteria. However, apart from P. aeruginosa, only Burkholderia pseudomallei and B. thailandensis contained a complete pqsA-E operon (termed hhqA-E). By introducing the B. pseudomallei hhqA and hhqE genes into P. aeruginosa pqsA and pqsE mutants, we show that they are functionally conserved and restore virulence factor and PQS production. B. pseudomallei, B. thailandensis, B. cenocepacia, and P. putida each produced 2-heptyl-4(1H)-quinolone (HHQ), but not PQS. Mutation of hhqA in B. pseudomallei resulted in the loss of AHQ production, altered colony morphology, and enhanced elastase production, which was reduced to parental levels by exogenous HHQ. These data reveal a role for AHQs in bacterial cell-to-cell communication beyond that seen in P. aeruginosa.     

12/10- and 11/13-mixed helices in alpha/gamma- and beta/gamma-hybrid peptides containing C-linked carbo-gamma-amino acids with alternating alpha- and beta-amino acids

New classes of alpha/gamma- and beta/gamma-hybrid peptides have been synthesized with novel 12/10- and 11/13-mixed helical patterns, respectively. The alpha/gamma-peptides were derived from the dipeptide repeats with alternating arrays of l-Ala and gamma-Caa((l)) (C-linked carbo-gamma-amino acid from d-mannose), which generated a new 12/10-mixed helix, for the first time, without a beta-amino acid. The beta/gamma-peptides made from an alternating arrangement of beta-Caa((x)) (C-linked carbo-beta-amino acid) and gamma-Caa((x)) (C-linked carbo-gamma-amino acid from d-xylose), on the other hand, resulted in an unprecedented 11/13-helix. The secondary structures in these peptides have been ascertained from detailed NMR studies, and CD spectroscopy and molecular dynamics investigations provided additional support for the structures derived.